Article

Engineered Reciprocal Chromosome Translocations Drive High Threshold, Reversible Population Replacement in Drosophila

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Abstract

Replacement of wild insect populations with transgene-bearing individuals unable to transmit disease or survive under specific environmental conditions using gene drive provides a self-perpetuating method of disease prevention. Mechanisms that require the gene drive element and linked cargo to exceed a high threshold frequency in order for spread to occur are attractive because they offer several points of control: they bring about local, but not global population replacement; and transgenes can be eliminated by reintroducing wildtypes into the population so as to drive the frequency of transgenes below the threshold frequency required for drive. Reciprocal chromosome translocations were proposed as a tool for bringing about high threshold population replacement in 1940 and 1968. However, translocations able to achieve this goal have only been reported once, in the spider mite Tetranychus urticae, a haplo-diploid species in which there is strong selection in haploid males for fit homozygotes. We report the creation of engineered translocation-bearing strains of Drosophila melanogaster, generated through targeted chromosomal breakage and homologous recombination. These strains drive high threshold population replacement in laboratory populations. While it remains to be shown that engineered translocations can bring about population replacement in wild populations, these observations suggest that further exploration of engineered translocations as a tool for controlled population replacement is warranted.

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... 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). ...
... In this chapter, we discuss considerations for field trials of gene drive systems, with a specific focus on confinement and reversibility criteria, and lessons learned from other genetics-based and biological control systems (Table 2.1). We pay special attention to reciprocal chromosomal translocations (Buchman et al. 2018), as an example of a threshold-dependent system that is confineable and reversible, and then extend our consideration to CRISPR-based homing gene drive systems and temporally self-limiting systems, such as split gene drive (Li et al. 2020), which could be used as confineable and reversible intermediate systems in a development pathway of homing-based systems. While these gene drive systems are yet to be trialed in the wild, lessons can be learned from trials of several varieties of sterile male mosquitoes, specifically, those sterilized through radiation (sterile insect technique, SIT), transfection with Wolbachia (incompatible insect technique, IIT) (Zheng et al. 2019;Crawford et al. 2020), and release of insects carrying a dominant lethal (RIDL) gene (Harris et al. 2011;Carvalho et al. 2015), as well as releases of Wolbachia-infected mosquitoes for population replacement (Hoffmann et al. 2011). ...
... This produces a threshold frequency of 50%, which increases in the presence of a fitness cost (Curtis 1968). Early attempts to generate translocations through radiation-induced mutagenesis were abandoned due to high associated fitness costs (Laven et al. 1972;Lorimer et al. 1972); however, interest has been reignited as site-specific translocations have recently been generated using CRISPR (Lekomtsev et al. 2016;Jiang et al. 2016), and translocations generated in D. melanogaster using endonucleases were recently shown to drive in laboratory experiments with a threshold frequency of~50% (Buchman et al. 2018). A recent modeling study suggests that translocations represent one of the best systems to implement in field trials due to their symmetrical threshold properties and strong confinement potential. ...
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.
... Here, we address this question as it applies to three broad categories of drive systems, all of which can likely be engineered more easily using CRISPR: a) threshold-dependent drives, b) threshold-independent drives, and c) temporally self-limiting drives. Threshold-dependent drives are distinct in the sense that they must exceed a critical frequency in the population in order to spread (4,5). These systems are well-suited for local confinement and may be eliminated from a population through being diluted below the threshold frequency. ...
... In the 1970's-80's, several attempts were made to generate reciprocal chromosomal translocations in mosquitoes to control wild populations; however these efforts were unsuccessful and the approach was ultimately abandoned (17,18). New efforts have refounded hope for this technique as precise chromosomal translocations were recently engineered using endonucleases and shown to drive in laboratory experiments with a threshold frequency of ~50% ( Figure 1A) (5). Site-specific chromosomal translocations have recently been engineered using CRISPR in several species, suggesting it is only a matter of time until CRIPSR-based translocation drives will be widely available (19)(20)(21). ...
... An optimal fitness cost may exist since there are competing demands -high fitness costs lead to rapid remediation following transient drive, but the maximum frequency that the system reaches in the population is compromised; while small fitness costs allow spread to higher maximum frequencies, but remediation from a population may take several years (22). Further complicating this, fitness costs are exceedingly difficult to quantify in the field, and even in the controlled environment of the laboratory, fitness costs have a tendency to change over time (4,5,9). It should also be noted that the spatial spread of temporally self-limiting drives is determined by how far the host species disperses while the drive system persists, and hence the degree of spatial dispersal is also determined by the fitness cost. ...
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The recent discovery of CRISPR and its application as a gene editing tool has enabled a range of gene drive systems to be engineered with much greater ease. In order for the benefits of this technology to be realized, drive systems must be developed that are capable of both spreading into populations to achieve their desired impact, and being recalled in the event of unwanted consequences or public disfavor. We review the performance of three broad categories of drive systems at achieving these goals - threshold-dependent drives, homing-based drive and remediation systems, and temporally self-limiting systems such as daisy-chain drives.
... Windbichler et al., 2007Windbichler et al., , 2008Windbichler et al., , 2011Hammond et al., 2016;Flores and O'Neill, 2018;Kyrou et al., 2018;. Likewise, increasing challenges associated with insecticide resistance in agricultural insect pests and the invasion of non-native insect species are driving the exploration of novel insect genetic control approaches, including engineered gene drives (Alphey, 2014;Dearden et al., 2017;Alphey and Bonsall, 2018;Buchman et al., 2018a;Lester and Beggs, 2019;Lester et al., 2020). ...
... However, the engineering of localised or transient gene drive systems (alone or in combination with other types of engineered gene drives) is mostly at the theoretical level, though a few have been tested under laboratory settings (e.g. Akbari et al., 2013Akbari et al., , 2014Reeves et al., 2014;Buchman et al., 2018a;Champer et al., 2020c;Terradas et al., 2020;Webster et al., 2020). While these approaches are reasonable for population modification strategies, modelling suggests that they may be less effective to suppress local target populations . ...
... Their introduction is also reversible, as releasing large numbers of wild-type individuals can push the gene drive below its threshold where over time it will become extinct in the population (Raban et al., 2020). Buchman et al. (2018a) created an engineered reciprocal chromosome translocations gene drive in D. melanogaster, using HEGs that carried a cargo/payload gene, and tested them under laboratory settings. The strains showed frequency-dependent spread in laboratory populations. ...
Article
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Advances in molecular and synthetic biology are enabling the engineering of gene drives in insects for disease vector/pest control. Engineered gene drives (that bias their own inheritance) can be designed either to suppress interbreeding target populations or modify them with a new genotype. Depending on the engineered gene drive system, theoretically, a genetic modification of interest could spread through target populations and persist indefinitely, or be restricted in its spread or persistence. While research on engineered gene drives and their applications in insects is advancing at a fast pace, it will take several years for technological developments to move to practical applications for deliberate release into the environment. Some gene drive modified insects (GDMIs) have been tested experimentally in the laboratory, but none has been assessed in small‐scale confined field trials or in open release trials as yet. There is concern that the deliberate release of GDMIs in the environment may have possible irreversible and unintended consequences. As a proactive measure, the European Food Safety Authority (EFSA) has been requested by the European Commission to review whether its previously published guidelines for the risk assessment of genetically modified animals (EFSA, 2012 and 2013), including insects (GMIs), are adequate and sufficient for GDMIs, primarily disease vectors, agricultural pests and invasive species, for deliberate release into the environment. Under this mandate, EFSA was not requested to develop risk assessment guidelines for GDMIs. In this Scientific Opinion, the Panel on Genetically Modified Organisms (GMO) concludes that EFSA's guidelines are adequate, but insufficient for the molecular characterisation (MC), environmental risk assessment (ERA) and post‐market environmental monitoring (PMEM) of GDMIs. While the MC,ERA and PMEM of GDMIs can build on the existing risk assessment framework for GMIs that do not contain engineered gene drives, there are specific areas where further guidance is needed for GDMIs.
... as other types of drives, such as homing based drives, it is inherently confinable and reversible which may be advantageous from a regulatory perspective [11][12][13][14] . Furthermore, confinable SPECIES underdominant systems are preferable to other underdominant systems, such as engineered translocations 16 , for local population replacement since they can tolerate higher fitness costs, spread more quickly, lead to less contamination of neighboring populations, and are more resilient to elimination due to immigration of wild types (Figs. 5 and 6). ...
... Regardless, it should be possible to implement SPECIES in organisms of medical or agricultural interest, such as mosquitoes. This becomes even more interesting considering the gene drive function of SPECIES provides greater control and confinement via threshold dependence 11,18,19 as well as a reversibility via WT 16 and protection from the evolution of resistance via multiplexing, features not found in all gene drives 11,14 . While release requirements for threshold-dependent systems are large due to fitness costs and may present logistical challenges, they are an order of magnitude less than those routinely carried out for insect suppression programs 20 . ...
... In Marshall and Hay 19 , population replacement and confinement dynamics are shown for: (1) extreme underdominance (the SPECIES system modeled here), (2) reciprocal chromosomal translocations 16 , (3) single-locus and two-locus engineered underdominance 32 , (4) Semele 33 , (5) inverse Medea 33 , and (6) Merea (Medea with a recessive antidote). A range of parameter values are compared for each gene drive system, including fitness cost (s, varied between 0 and 30%) and migration rate (m, varied between 0 and 10% per individual per generation for both the source and two-population models) 16 . ...
Article
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Engineered reproductive species barriers are useful for impeding gene flow and driving desirable genes into wild populations in a reversible threshold-dependent manner. However, methods to generate synthetic barriers have not been developed in advanced eukaryotes. To overcome this challenge, we engineered SPECIES (Synthetic Postzygotic barriers Exploiting CRISPR-based Incompatibilities for Engineering Species) to generate postzygotic reproductive barriers. Using this approach, we engineer multiple reproductively isolated SPECIES and demonstrate their threshold-dependent gene drive capabilities in D. melanogaster. Given the near-universal functionality of CRISPR tools, this approach should be portable to many species, including insect disease vectors in which confinable gene drives could be of great practical utility.
... Each system has its own strengths and weaknesses and could be suited to a different situation. In this paper, we theoretically explore the potential for two recently engineered thresholddependent gene drive systems to achieve localized and reversible population modification in structured populations-reciprocal chromosomal translocations [14] and a toxin-antidote-based system known as UD MEL [15]. ...
... When translocation heterozygotes mate, several crosses result in unbalanced genotypes and hence unviable offspring, resulting in a heterozygote reproductive disadvantage. This results in bistable, threshold-dependent population dynamics, confirmed in laboratory drive experiments [14]. The inheritance patterns produced by the UD MEL system are depicted in Fig. 1b. ...
... The use of translocations for transforming pest populations was initially suggested by Serebrovskii [43] and later Curtis [44] for the introduction of diseaserefractory genes into mosquito populations. A number of models have been proposed to describe their spread through randomly mating populations [14,16,45,46]; however, with one recent exception addressing spatial structure [18], these have largely ignored insect life history and mating structure. Such models suggest that the translocation need only exceed a population frequency of 50%, in the absence of a fitness cost associated with the translocation, to spread to fixation in a population, which could conceivably be achieved through a single seeding release round. ...
Article
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The discovery of CRISPR-based gene editing and its application to homing-based gene drive systems 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. Gene drive systems that display threshold-dependent behavior could potentially be used during the trial phase of this technology, or when localized control is otherwise desired, as simple models predict them to spread into partially isolated populations in a confineable manner, and to be reversible through releases of wild-type organisms. Here, we model hypothetical releases of two recently engineered threshold-dependent gene drive systems-reciprocal chromosomal translocations and a form of toxin-antidote-based underdominance known as UD MEL-to explore their ability to be confined and remediated. We simulate releases of Aedes aegypti, the mosquito vector of dengue, Zika, and other arboviruses, in Yorkeys Knob, a suburb of Cairns, Australia, where previous biological control interventions have been undertaken on this species. We monitor spread to the neighboring suburb of Trinity Park to assess confinement. Results suggest that translocations could be introduced on a suburban scale, and remediated through releases of non-disease-transmitting male mosquitoes with release sizes on the scale of what has been previously implemented. UD MEL requires fewer releases to introduce, but more releases to remediate, including of females capable of disease transmission. Both systems are expected to be confineable to the release site; however, spillover of translocations into neighboring populations is less likely. Our analysis supports the use of translocations as a threshold-dependent drive system capable of spreading disease-refractory genes into Ae. aegypti populations in a confineable and reversible manner. It also highlights increased release requirements when incorporating life history and population structure into models. As the technology nears implementation, further ecological work will be essential to enhance model predictions in preparation for field trials.
... The meiotic drive, sex-ratio distortion, and replicative transposition are typical examples of naturally occurring gene-drive elements (Ågren, 2016). The synthetic Medea drive system (Chen et al., 2007;Akbari et al., 2014), engineered underdominance systems (Buchman et al., 2018), and homing endonuclease gene-drive (HEGD) (Marshall and Hay, 2012;Esvelt et al., 2014;Champer et al., 2016) are included in synthetic gene-drive systems. The development of HEGD is accelerated by discovering the CRISPR/Cas9 system (Jinek et al., 2012;Cong et al., 2013;Mali et al., 2013). ...
... The CCGD has recently been used to control the pest population through a population-modification and population-suppression drives. In a population-modification drive, the release of transgenic mosquitoes in which the CCGD contains effector genes inhibiting the mosquito pathogen transmission results in the replacement of diseasesensitive mosquitoes with disease-resistant mosquitoes, which decreases the pathogen transmission (Buchman et al., 2018;Buchman et al., 2020). In the population-suppression drive, the homing-based gene-drive is developed to target the conserved sex-specific genes (Kyrou et al., 2018). ...
Article
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The gene-drive system can ensure that desirable traits are transmitted to the progeny more than the normal Mendelian segregation. The clustered regularly interspersed palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) mediated gene-drive system has been demonstrated in dipteran insect species, including Drosophila and Anopheles, not yet in other insect species. Here, we have developed a single CRISPR/Cas9-mediated gene-drive construct for Plutella xylostella, a highly-destructive lepidopteran pest of cruciferous crops. The gene-drive construct was developed containing a Cas9 gene, a marker gene (EGFP) and a gRNA sequence targeting the phenotypic marker gene (Pxyellow) and site-specifically inserted into the P. xylostella genome. This homing-based gene-drive copied ∼12 kb of a fragment containing Cas9 gene, gRNA, and EGFP gene along with their promoters to the target site. Overall, 6.67%–12.59% gene-drive efficiency due to homology-directed repair (HDR), and 80.93%–86.77% resistant-allele formation due to non-homologous-end joining (NHEJ) were observed. Furthermore, the transgenic progeny derived from male parents showed a higher gene-drive efficiency compared with transgenic progeny derived from female parents. This study demonstrates the feasibility of the CRISPR/Cas9-mediated gene-drive construct in P. xylostella that inherits the desired traits to the progeny. The finding of this study provides a foundation to develop an effective CRISPR/Cas9-mediated gene-drive system for pest control.
... 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. A particular characteristic of many of these drives is that they are expected to behave in a frequencydependent manner; transgenic insects are predicted to spread into a population and eventually reach fixation only if seeded above a critical threshold, which largely depends on the relative fitness of the engineered insects as well as the construct design [78]. ...
... 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. A particular characteristic of many of these drives is that they are expected to behave in a frequency-dependent manner; transgenic insects are predicted to spread into a population and eventually reach fixation only if seeded above a critical threshold, which largely depends on the relative fitness of the engineered insects as well as the construct design [78]. ...
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.
... The induction of several DNA double-strand breaks simultaneously can lead to complex rearrangements in a genome by joining unrelated break ends by nonhomologous end-joining (NHEJ), thus, double-strand breaks on two distinct chromosomes can lead to reciprocal translocations. Chromosome translocations have been successfully achieved via CRISPR/Cas9 in various cell lines [142][143][144] and model organisms Caenorhabditis elegans [145], D. melanogaster [146] and Arabidopsis thaliana [147]. Establishing insect strains with CRISPR/Cas9 site induced translocations is thought to have potential beyond these model systems, provided adequate reference genome sequences are available to aid with experimental design [146,148]. ...
... Chromosome translocations have been successfully achieved via CRISPR/Cas9 in various cell lines [142][143][144] and model organisms Caenorhabditis elegans [145], D. melanogaster [146] and Arabidopsis thaliana [147]. Establishing insect strains with CRISPR/Cas9 site induced translocations is thought to have potential beyond these model systems, provided adequate reference genome sequences are available to aid with experimental design [146,148]. Whether developed with CRISPR/Cas9 or classical approaches, chromosomal translocations can be stabilized with inversions to minimize unwanted recombination events. However, inversions can impact strain productivity when recombination occurs through the creating imbalanced gametes which are unable to develop [30], representing yet another challenge for maintaining stable GSS colonies for SIT. ...
Article
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A major obstacle of sterile insect technique (SIT) programs is the availability of robust sex-separation systems for conditional removal of females. Sterilized male-only releases improve SIT efficiency and cost-effectiveness for agricultural pests, whereas it is critical to remove female disease-vector pests prior to release as they maintain the capacity to transmit disease. Some of the most successful Genetic Sexing Strains (GSS) reared and released for SIT control were developed for Mediterranean fruit fly (Medfly), Ceratitis capitata, and carry a temperature sensitive lethal (tsl) mutation that eliminates female but not male embryos when heat treated. The Medfly tsl mutation was generated by random mutagenesis and the genetic mechanism causing this valuable heat sensitive phenotype remains unknown. Conditional temperature sensitive lethal mutations have also been developed using random mutagenesis in the insect model, Drosophila melanogaster, and were used for some of the founding genetic research published in the fields of neuro- and developmental biology. Here we review mutations in select D. melanogaster genes shibire, Notch, RNA polymerase II 215kDa, pale, transformer-2, Dsor1 and CK2α that cause temperature sensitive phenotypes. Precise introduction of orthologous point mutations in pest insect species with CRISPR/Cas9 genome editing technology holds potential to establish GSSs with embryonic lethality to improve and advance SIT pest control.
... 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]. ...
... 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]. Much work is being put in to understanding the formation of alleles resistant to CRISPR/Cas9 cleavage [20,21,[26][27][28][29] and to increasing gene drive efficiencies overall [22,27,28,[30][31][32]. ...
Article
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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.
... Certain types of drive (e.g. translocations) are much less likely to face resistance, but may spread more slowly than drives that bias segregation (Buchman, Ivy, et al., 2018;Champer et al., 2016). Additionally, population suppression drives will face considerably stronger evolutionary pressures in terms of resistance than replacement drives (Eckhoff, Wenger, Godfray, & Burt, 2017;KaramiNejadRanjbar et al., 2018;Prowse et al., 2017). ...
... There are several design strategies that may help prevent target-site resistance. Firstly, targets in essential and/ or highly conserved sequences/genes may be less tolerant of sequence variability/polymorphism and thus less likely to harbour pre-existing resistance alleles, or to tolerate novel mutational variation(Buchman, Ivy, Marshall, Akbari, & Hay, 2018). For homing-based systems, gene drives could home into genes that are essential, so that incorrect homing events (e.g. ...
Article
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Scientists are rapidly developing synthetic gene drive elements intended for release into natural populations. These are intended to control or eradicate disease vectors and pests, or to spread useful traits through wild populations for disease control or conservation purposes. However, a crucial problem for gene drives is the evolution of resistance against them, preventing their spread. Understanding the mechanisms by which populations might evolve resistance is essential for engineering effective gene drive systems. This review summarizes our current knowledge of drive resistance in both natural and synthetic gene drives. We explore how insights from naturally occurring and synthetic drive systems can be integrated to improve the design of gene drives, better predict the outcome of releases and understand genomic conflict in general.
... When released into a population below that critical frequency, they should be lost over time. Since spreading dynamics depend on local population genetic frequencies, threshold drives could be easier to keep localized as long as there is limited gene flow between separate populations (Marshall and Hay 2012;Akbari et al. 2013;Buchman et al. 2018;Marshall and Akbari 2018). If this critical threshold frequency is substantially above 0, a small number of individuals carrying the gene drive dispersing to a new population should be unlikely to spread through its new population. ...
... If this critical threshold frequency is substantially above 0, a small number of individuals carrying the gene drive dispersing to a new population should be unlikely to spread through its new population. Moreover, if managers want to reverse the spread of the gene drive or remove a gene drive from any location, they could release enough wild-type individuals to push the population back below the critical threshold frequency (Akbari et al. 2013;Buchman et al. 2018). Importantly, critical thresholds depend partially on the gene drive's molecular construction, but also on the fitness of the individuals carrying the gene drive (Ward et al. 2010;Marshall and Hay 2012). ...
Article
Gene drive technology could allow the intentional spread of a desired gene throughout an entire wild population in relatively few generations. However, there are major concerns that gene drives could either fail to spread or spread without restraint beyond the targeted population. One potential solution is to use more localized threshold-dependent drives, which only spread when they are released in a population above a critical frequency. However, under certain conditions, small changes in gene drive fitness could lead to divergent outcomes in spreading behavior. In the face of ecological uncertainty, the inability to estimate gene drive fitness in a real-world context could prove problematic because gene drives designed to be localized could spread to fixation in neighboring populations if ecological conditions unexpectedly favor the gene drive. This perspective offers guidance to developers and managers because navigating gene drive spread and controllability could be risky without detailed knowledge of ecological contexts. Full text available: https://academic.oup.com/bioscience/advance-article-abstract/doi/10.1093/biosci/biz098/5559621
... Various types of toxin-antidote drives have also been modeled [18][19][20][21][22][23][24]. Some of these have been proven to be capable of spreading in Drosophila experiments [25][26][27][28][29][30][31]. However, more research is needed to find suitable promoters and target sites and to successfully construct these new types of drives with desired properties in target species. ...
Article
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Wolbachia are maternally-inherited bacteria, which can spread rapidly in populations by manipulating reproduction. cifA and cifB are genes found in Wolbachia phage that are responsible for cytoplasmic incompatibility, the most common type of Wolbachia reproductive interference. In this phenomenon, no viable offspring are produced when a male with both cifA and cifB (or just cifB in some systems) mates with a female lacking cifA . Utilizing this feature, we propose new types of toxin-antidote gene drives that can be constructed with only these two genes in an insect genome, instead of the whole Wolbachia bacteria. By using both mathematical and simulation models, we found that a drive containing cifA and cifB together creates a confined drive with a moderate to high introduction threshold. When introduced separately, they act as a self-limiting drive. We observed that the performance of these drives is substantially influenced by various ecological parameters and drive characteristics. Extending our models to continuous space, we found that the drive individual release distribution has a critical impact on drive persistence. Our results suggest that these new types of drives based on Wolbachia transgenes are safe and flexible candidates for genetic modification of populations.
... Several options exist for confined gene drives 3,5 , including Medea 9 , haploinsufficient underdominance 10 , chromosomal rearrangements 11,12 , incompatibility underdominance 13 , and Wolbachia cytoplasmic incompatibility drive 14 . Some of the latest and most promising confined drive designs are based on CRISPR nucleases and can avoid functional resistance alleles by use of multiplexed gRNAs. ...
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New types of gene drives promise to provide increased flexibility, offering many options for confined modification or suppression of target populations. Among the most promising are CRISPR toxin-antidote gene drives, which disrupt essential wild-type genes by targeting them with Cas9/gRNA, resulting in their removal. This increases the frequency of the drive in the population. All these drives, plus homing modification rescue drives, rely on having an effective rescue element, which consists of a recoded version of the target gene. This rescue element can be at the same site as the target gene, which maximizes the chance of efficient rescue, or at a distant site, which allows some other useful options, such as easily disrupting another essential gene or achieving greater confinement. Previously, we developed a homing rescue drive targeting a haplolethal gene and a toxin-antidote drive targeting an essential but haplosufficient gene. These successful drives had functional rescue elements but suboptimal drive efficiency. Here, we attempted to construct new toxin-antidote drives targeting these genes with a distant-site configuration from three different loci. We found that use of additional gRNAs increased cut rates to nearly 100%. However, all distant-site rescue elements failed for both haplolethal and haplosufficient target genes. Furthermore, one rescue element with a minimally recoded rescue element was used as a template for homology-directed repair for the target gene on a different chromosomal arm, resulting in the formation of functional resistance alleles at high frequency. Together, these results can inform the design of future CRISPR-based toxin-antidote gene drives.
... 最近, 基因驱动(gene drive)这种超孟德尔式遗传 (super-Mendelian inheritance)方式因其可使特定遗传 元件快速地以高频率(可达95%以上) [ [12,13] . [14] ; 以及在发生染色体易位的黑腹果蝇(Drosophila melanogaster)中, 后代杂合子适合度低于纯合子 适合度的显性不足效应(underdominance) [15] 等. 这类基 于选择的基因驱动现象从遗传模式上看仍为孟德尔遗 传(Mendelian inheritance), 故难以使种群中特定遗传元 件的频率在短世代内到达高峰值. 此外, 由于受正选择 青睐的遗传元件的来源及其在物种间通用性等方面的 限制, 并不容易通过分子操作来实现此类基因驱动 [16] . ...
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Gene drive refers to the phenomenon that specific genes or genetic elements are passed from parent to offspring in the form of super-Mendelian inheritance. In recent years, based on the genetic characteristics of gene drive and the theoretical basis of their molecular mechanisms, and supported by CRISPR/Cas9 gene-editing system, gene-driven genetic control technology has become an advanced research hotspot in the field of basic mosquito biology and genetic control. And there have emerged some practical and effective achievements that take into account ecological stability. This paper reviews the basic principles of gene drive, CRISPR/Cas9-mediated and HDR-type drive technology strategies, improved strategies to reduce gene-driven resistance and potential risks, and simulation analysis of gene drive. It is hoped to provide a reference for the development of a gene-driven mosquito genetic control technology system that takes both high efficiency and safety into consideration.
... Various types of toxinantidote drives have also been modeled [18][19][20][21][22][23][24]. Some of these have been proven to be capable of spreading in Drosophila experiments [25][26][27][28][29][30][31]. However, more research is needed to find suitable promoters and target sites and to successfully construct these new types of drives with desired properties in target species. ...
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Wolbachia is a maternally-inherited bacteria, which can spread rapidly in populations by manipulating reproduction. CifA and CifB are genes found in Wolbachia phage that are responsible for cytoplasmic incompatibility, the most common type of Wolbachia reproductive interference. In this phenomenon, no viable offspring are produced when a male with both CifA and CifB (or just CifB in an alternative mechanism) mates with a female lacking CifA. Utilizing this feature, we propose new types of toxin-antidote gene drives that can be constructed with these genes. By using both mathematical and simulation models, we found that a drive containing CifA and CifB together create a confined drive with a moderate to high introduction threshold. When introduced separately, they act as a self-limiting drive. We observed that the performance of these drives is substantially influenced by various ecological parameters and drive characteristics. Extending our models to continuous space, we found that the drive individual release distribution has a critical impact on drive persistence. Our results suggest that these new types of drives based on Wolbachia transgenes are safe and flexible candidates for genetic modification of populations.
... Afterwards, a similar drive system was established in the spider mite Tetranychus urticae, a haplodiploid species in which there is a substantial selection in haploid males for fit homozygotes [56]. Recently, Buchman et al. [57] reported the generation of engineered translocation-bearing strains of D. melanogaster through targeted chromosomal breakage, using a site-specific nuclease, followed by homologous recombination. Findings from this study revealed high threshold population replacement in laboratory populations, while it remains to be explored in wild populations. ...
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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.
... Toxin-antidote drives Several older forms of toxin-antidote gene drives have been studied, though recent progress has been slow. Chromosomal rearrangements with introduction frequencies of 50% have been engineered in A. aegypti [84], Anopheles [85][86][87], and flies [88]; however, difficulty of engineering and often high fitness costs have prevented progress with this method in mosquitoes for the past few decades. 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]. ...
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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.
... Several new techniques for genetic biocontrol have been shown effective in proof-of-concept experiments (Alphey and Bonsall, 2014;Maselko et al., 2020;Buchman et al., 2021;Buchman et al., 2018;Akbari et al., 2014;Champer et al., 2020;Gantz and Bier, 2015;Terradas et al., 2021;Windbichler et al., 2011). Combination approaches that bring together two or more distinct techniques can exhibit synergistic behaviors that are attractive for genetic biocontrol applications. ...
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Engineered Genetic Incompatibility (EGI) is a method to create species-like barriers to sexual reproduction. It has applications in pest control that mimic Sterile Insect Technique when only EGI males are released. This can be facilitated by introducing conditional female-lethality to EGI strains to generate a sex-sorting incompatible male system (SSIMS). Here, we demonstrate a proof of concept by combining tetracycline-controlled female lethality constructs with a pyramus -targeting EGI line in the model insect Drosophila melanogaster . We show that both functions (incompatibility and sex-sorting) are robustly maintained in the SSIMS line and that this approach is effective for population suppression in cage experiments. Further we show that SSIMS males remain competitive with wild-type males for reproduction with wild-type females, including at the level of sperm competition.
... Gene drive is a novel method that involves the inheritance of specific traits from one generation to the next at rates higher than the 50% chance afforded through Mendelian inheritance in heterozygotes, and gives certain genes a substantially higher or lower probability of inheritance and thereby alters the frequency of such genes in the population. A gene that alters the fertility or survival of the target species could thereby alter the species population size, depending on the species and the drive system applied (Beaghton et al., 2017;Buchman et al., 2018;Burt & Deredec, 2018;Gantz et al., 2015;Hammond & Galizi, 2017;Marshall et al., 2011;North et al., 2019North et al., , 2020. Given rising resistance to existing insecticides and antimalarial drugs (Bhagavathula et al., 2016;Bull et al., 2019;Mnzava et al., 2015;Protopopoff et al., 2018;WHO, 2014), gene drive mosquitoes might hold great potential to accelerate and achieve lasting gains in malaria control (Committee on Gene Drive Research in Non-Human Organisms, 2016). ...
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Malaria elimination will be challenging in countries that currently continue to bear high malaria burden. Sex-ratio distorting gene drives, such as driving-Y, could play a role in an integrated elimination strategy if they can effectively suppress vector populations. Using a spatially explicit, agent-based model of malaria transmission in eight provinces spanning the range of transmission intensities across the Democratic Republic of the Congo, we predict the impact and cost-effectiveness of integrating driving-Y gene drive mosquitoes in malaria elimination strategies that include existing interventions such as insecticide-treated nets and case management of symptomatic malaria. Gene drive mosquitoes could eliminate malaria and were the most cost-effective intervention overall if the drive component was highly effective with at least 95% X-shredder efficiency at relatively low fertility cost, and associated cost of deployment below 7.17 $int per person per year. Suppression gene drive could be a cost-effective supplemental intervention for malaria elimination, but tight constraints on drive effectiveness and cost ceilings may limit its feasibility.
... This Review focuses on the latter low-threshold gene drives in insects as they arguably hold the greatest promise for impacting disease transmission on continental scales and because optimized second-generation drives have been developed over the past 5 years. Various high-threshold systems, with diverse applications on more local scales, are reviewed elsewhere, including second-generation so-called underdominant chromosome translocation systems 22 and other strategies such as Medea toxinantitoxin arrangements [23][24][25][26][27][28][29] , Cleave and Rescue 30,31 or TARE systems 32 , in which an essential gene is targeted for inactivation and rescued by a recoded transgene inserted elsewhere in the genome, or reproductive symbionts/ parasites such as Wolbachia [33][34][35] . ...
Article
Gene drives are selfish genetic elements that are transmitted to progeny at super-Mendelian (>50%) frequencies. Recently developed CRISPR–Cas9-based gene-drive systems are highly efficient in laboratory settings, offering the potential to reduce the prevalence of vector-borne diseases, crop pests and non-native invasive species. However, concerns have been raised regarding the potential unintended impacts of gene-drive systems. This Review summarizes the phenomenal progress in this field, focusing on optimal design features for full-drive elements (drives with linked Cas9 and guide RNA components) that either suppress target mosquito populations or modify them to prevent pathogen transmission, allelic drives for updating genetic elements, mitigating strategies including trans-complementing split-drives and genetic neutralizing elements, and the adaptation of drive technology to other organisms. These scientific advances, combined with ethical and social considerations, will facilitate the transparent and responsible advancement of these technologies towards field implementation. In this Review, Ethan Bier discusses how several impactful technical advancements, particularly involving CRISPR-based methods, are providing a diverse toolkit of gene-drive systems for the control of populations such as insect vectors of disease.
... While the argument could conceptually be extended to any drive with an introduction threshold resulting from such costs, the uncertainty and variability associated with such costs make them extremely challenging to evaluate before release, and the corresponding frequency thresholds in natural populations are both spatially and temporally variable and dependent on environmental factors (Backus & Delborne, 2019). To clarify, we will focus in this paragraph on gene drives that are threshold-dependent even in the absence of fitness costs, such as engineered underdominance (Figure 1b,c), which comes in many possible forms (Davis et al., 2001;Magori, 2005), or translocations, natural or engineered (Buchman, Ivy, et al., 2018;Curtis, 1968;Gould & Schliekelman, 2004). ...
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Recent advances in gene editing technologies have opened new avenues for genetic pest control strategies, in particular around the use of gene drives to suppress or modify pest populations. Significant uncertainty, however, surrounds the applicability of these strategies to novel target species, their efficacy in natural populations, and their eventual safety and acceptability as control methods. In this article we identify issues associated with the potential use of gene drives in agricultural systems, to control pests and diseases that impose a significant cost to agriculture around the world. We first review the need for innovative approaches, and provide an overview of the most relevant biological and ecological traits of agricultural pests that could impact the outcome of gene drive approaches. We then describe the specific challenges associated with using gene drives in agricultural systems, as well as the opportunities that these environments may offer, focusing in particular on the advantages of high-threshold gene drives. Overall we aim to provide a comprehensive view of the potential opportunities and the remaining uncertainties around the use of gene drives in agricultural systems.
... 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. ...
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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.
... While current research is investigating the development of engineered GDs in insect populations and deploying them, it will take many years before they can be applied to practical disease vector/pest management. At present, some GDMIs are either in development or have been tested experimentally in the laboratory, often with multigenerational data and model simulations [17,19,20,27,28,33,35,39,43,45,46,[53][54][55][56][57][58][59][60][61][62][63][64][65][66][67]. However, no "contemporary" GDMIs have been assessed in small-scale physically and/or ecologically confined field trials, or open release trials [5,8,10,15,68]. ...
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Potential future application of engineered gene drives (GDs), which bias their own inheritance and can spread genetic modifications in wild target populations, has sparked both enthusiasm and concern. Engineered GDs in insects could potentially be used to address long-standing challenges in control of disease vectors, agricultural pests and invasive species, or help to rescue endangered species, and thus provide important public benefits. However, there are concerns that the deliberate environmental release of GD modified insects may pose different or new harms to animal and human health and the wider environment, and raise novel challenges for risk assessment. Risk assessors, risk managers, developers, potential applicants and other stakeholders at many levels are currently discussing whether there is a need to develop new or additional risk assessment guidance for the environmental release of GD modified organisms, including insects. Developing new or additional guidance that is useful and practical is a challenge, especially at an international level, as risk assessors, risk managers and many other stakeholders have different, often contrasting, opinions and perspectives toward the environmental release of GD modified organisms, and on the adequacy of current risk assessment frameworks for such organisms. Here, we offer recommendations to overcome some of the challenges associated with the potential future development of new or additional risk assessment guidance for GD modified insects and provide considerations on areas where further risk assessment guidance may be required.
... Several new techniques for genetic biocontrol have been shown effective in proof-of-concept experiments (13,15,19,(25)(26)(27)(28)(29)(30). Combination approaches that bring together two or more distinct techniques can exhibit synergistic behaviors that are attractive for genetic biocontrol applications. ...
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Engineered Genetic Incompatibility (EGI) is a method to create species-like barriers to sexual reproduction. It has applications in pest control that mimic Sterile Insect Technique when only EGI males are released. This can be facilitated by introducing conditional female-lethality to EGI strains to generate a sex-sorting incompatible male system (SSIMS). Here we demonstrate a proof of concept by combining tetracycline-controlled female lethality constructs with a pyramus -targeting EGI line in the model insect Drosophila melanogaster . We show that both functions (incompatibility and sex-sorting) are robustly maintained in the SSIMS line and that this approach is effective for population suppression in cage experiments. Further we show that SSIMS males remain competitive with wild-type males for reproduction with wild-type females, including at the level of sperm competition.
... Here, a male-linked transgene (located on the Y-chromosome) could, for example, target a gene required for female viability, or include a gene whose expression results in non-viability of female offspring (Backus and Gross 2016;Godwin et al. 1914) such that all female progeny of a transgenic male are non-viable, while all male progeny are viable and transgenic. Readers may find (Chen et al. 2007) and engineered reciprocal translocations (Buchman et al. 2018) can very broadly be described as 'toxin-antidote' drives in that they operate through various means to create a rescuable genetic load in a target population. Orthogonal mechanisms to induce drive spread include the 'cut, copy and paste' mechanism of nuclease-based homing drives where a homologous chromosome is cut by the drive allele, with that drive allele being copied across into the cut site during the chromosomal repair process (known as 'homing') (Burt 2003). ...
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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.
... Several molecular strategies have been proposed to limit gene drive spread including physical separation of gene drive components ("split drive"; DiCarlo et al. 2015) or gene drives that only function above a certain population frequency threshold (Buchman et al. 2018;Leftwich et al. 2018). Engineering the system such that there is a marker gene could also be useful for monitoring containment (Beech et al. 2009). ...
Article
Vertebrate wildlife damage management relates to developing and employing methods to mitigate against damage caused by wildlife in the areas of food production, property damage, and animal or human health and safety. Of the many management tools available, chemical methods (e.g., toxicants) draw the most attention owing to issues related to environmental burden, species specificity, and humaneness. Research and development focusing on RNA interference and gene drives may be able to address the technical aspects of performance goals. However, there remain many questions about regulation, environmental risk, and societal acceptance for these emerging biological technologies. Here we focus on the development and use of these biological technologies for use in vertebrate pest management and conservation (e.g., management of wildlife diseases). We then discuss the regulatory framework and challenges these technologies present and conclude with a discussion on factors to consider for enabling these technologies for pest management and conservation applications under a commercially applied framework.
... Several molecular strategies have been proposed to limit gene drive spread including physical separation of gene drive components ("split drive"; DiCarlo et al. 2015) or gene drives that only function above a certain population frequency threshold (Buchman et al. 2018;Leftwich et al. 2018). Engineering the system such that there is a marker gene could also be useful for monitoring containment (Beech et al. 2009). ...
Chapter
Vertebrate wildlife damage management relates to developing and employing methods to mitigate against damage caused by wildlife in the areas of food production, property damage, and animal or human health and safety. Of the many management tools available, chemical methods (e.g., toxicants) draw the most attention owing to issues related to environmental burden, species specificity, and humaneness. Research and development focusing on RNA interference and gene drives may be able to address the technical aspects of performance goals. However, there remain many questions about regulation, environmental risk, and societal acceptance for these emerging biological technologies. Here we focus on the development and use of these biological technologies for use in vertebrate pest management and conservation (e.g., management of wildlife diseases). We then discuss the regulatory framework and challenges these technologies present and conclude with a discussion on factors to consider for enabling these technologies for pest management and conservation applications under a commercially applied framework.
... A number of approaches to spreading traits through populations (population replacement/alteration/modification) in ways that are self-sustaining, by linking them with genetic elements that mediate drive, have been proposed (5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22) . Several of these, Medea (9,23) , UD mel (15) , engineered translocations (24) , and ClvR ( Cleave and Rescue ) selfish genetic elements (25) , have been implemented and shown to spread to transgene fixation in otherwise wildtype Drosophila . Sustained modification of a wildtype mosquito population using a homing based strategy, resulting in population suppression, has also been reported (26) . ...
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Gene drive-based strategies for modifying populations face the problem that genes encoding cargo and the drive mechanism are subject to separation, mutational inactivation, and loss of efficacy. Resilience, an ability to respond to these eventualities in ways that restore population modification with functional genes is needed for long-term success. Here we show that resilience can be achieved through cycles of population modification with “ Cleave and Rescue” ( ClvR ) selfish genetic elements. ClvR comprises a DNA sequence-modifying enzyme such as Cas9/gRNAs that disrupts endogenous versions of an essential gene, and a recoded version of the essential gene resistant to cleavage. ClvR spreads by creating conditions in which those lacking ClvR die because they lack functional versions of the essential gene. Cycles of modification can in principal be carried out if two ClvR elements targeting different essential genes are located at the same genomic position, and one of them, ClvR ⁿ⁺¹ , carries a Rescue transgene from an earlier element, ClvR ⁿ . ClvR ⁿ⁺¹ should spread within a population of ClvR ⁿ , while also bringing about a decrease in its frequency. To test this hypothesis we first show that multiple ClvR s, each targeting a different essential gene, function when located at a common chromosomal position in Drosophila . We then show that when several of these also carry the Rescue from a different ClvR , they spread to transgene fixation in populations fixed for the latter, and at its expense. Therefore, genetic modifications of populations can be overwritten with new content, providing an ongoing point of control. Significance Gene drive can spread beneficial traits through populations, but will never be a one-shot project in which one genetic element provides all desired modifications, for an indefinitely long time. Here we show that gene drive mediated population modification in Drosophila can be overwritten with new content while eliminating old, using Cleave and Rescue ( ClvR ) selfish genetic elements. The ability to carry out cycles of modification that create and then leave behind a minimal genetic footprint while entering and exiting a population provides important points of control. It makes possible the replacement of broken elements, upgrades with new elements that better carry out their tasks and/or provide new functions, all while promoting the removal of modifications no longer needed.
... If threshold drives were to be used for eradication or suppression, they would face an even stronger challenge when spreading across multiple populations, assuming they can spread within a population in the first place. This theoretical inability of threshold drives to spread across populations, or in other words, the ability to remain localized to a population, is attractive when spread across multiple populations is undesirable (Buchman et al. 2018a, Curtis & Robinson 1971, Davis et al. 2001, Dhole et al. 2018, Magori & Gould 2006, Marshall 2010, Marshall & Hay 2012a, Natl. Acad. ...
Article
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The spread of synthetic gene drives is often discussed in the context of panmictic populations connected by gene flow and described with simple deterministic models. Under such assumptions, an entire species could be altered by releasing a single individual carrying an invasive gene drive, such as a standard homing drive. While this remains a theoretical possibility, gene drive spread in natural populations is more complex and merits a more realistic assessment. The fate of any gene drive released in a population would be inextricably linked to the population's ecology. Given the uncertainty often involved in ecological assessment of natural populations, understanding the sensitivity of gene drive spread to important ecological factors is critical. Here we review how different forms of density dependence, spatial heterogeneity, and mating behaviors can impact the spread of self-sustaining gene drives. We highlight specific aspects of gene drive dynamics and the target populations that need further research. Expected final online publication date for the Annual Review of Ecology, Evolution, and Systematics, Volume 51 is November 2, 2020. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.
... If threshold drives were to be used for eradication or suppression, they would face an even stronger challenge when spreading across multiple populations, assuming they can spread within a population in the first place. This theoretical inability of threshold drives to spread across populations, or in other words, the ability to remain localized to a population is attractive when spread across multiple populations is undesirable (Buchman et al. 2018b;Curtis & Robinson 1971;Davis et al. 2001;Dhole et al. 2018;Magori & Gould 2006;Marshall 2010;Marshall & Hay 2012b; National Academies of Sciences and Medicine 2016). ...
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The spread of synthetic gene drives is often discussed in the context of panmictic populations connected by gene flow and described with simple deterministic models. Under such assumptions, an entire species could be altered by releasing a single individual carrying an invasive gene drive, such as a standard homing drive. While this remains a theoretical possibility, gene drive spread in natural populations is more complex and merits a more realistic assessment. The fate of any gene drive released in a population would be inextricably linked to the ecology of the population. Given the uncertainty often involved in ecological assessment of natural populations, understanding the sensitivity of gene drive spread to important ecological factors is critical. Here we review how different forms of density-dependence, spatial heterogeneity and mating behaviors can impact the spread of self-sustaining gene drives. We highlight specific aspects of gene drive dynamics and the target populations that need further research.
... The earliest systems involved the formation of reciprocal chromosomal trans-locations (RCT). [9][10][11][12] More recently, toxin-antidote systems at a single genomic locus (1L1T) have been developed, 13 as well as two-allele toxin-antidote systems at a single locus (1L2T) or two genetically unlinked loci (2L2T). 14 The expected population dynamics of these systems have been examined in several modeling studies, which typically focused on panmictic population models and extensions of such models to two or more discrete panmictic demes linked by migration. ...
Article
Underdominance systems can quickly spread through a population, but only when introduced in considerable numbers. This promises a gene drive mechanism that is less invasive than homing drives, potentially enabling new approaches in the fight against vector-borne diseases. If regional confinement can indeed be achieved, the decision-making process for a release would likely be much simpler compared to other, more invasive types of drives. The capacity of underdominance gene drive systems to spread in a target population without invading other populations is typically assessed via network models of panmictic demes linked by migration. However, it remains less clear how such systems would behave in more realistic population models where organisms move over a continuous landscape. Here, we use individual-based simulations to study the dynamics of several proposed underdominance systems in continuous-space. We find that all these systems can fail to persist in such environments, even after an initially successful establishment in the release area, confirming previous theoretical results from diffusion theory. At the same time, we find that a two-locus two-toxin-antidote system can invade connected demes through a narrow migration corridor. This suggests that the parameter space where underdominance systems can establish and persist in a release area while at the same time remaining confined to that area could be quite limited, depending on how a population is spatially structured. Overall, these results indicate that realistic spatial context must be considered when assessing strategies for the deployment of underdominance drives.
... A number of approaches to spreading traits through populations (population replacement/alteration/modification) in ways that are self-sustaining, by linking them with genetic elements that mediate drive, have been proposed (5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22) . Several of these, Medea (9,23) , UD mel (15) , engineered translocations (24) , and ClvR ( Cleave and Rescue ) selfish genetic elements (25) , have been implemented and shown to spread to transgene fixation in otherwise wildtype Drosophila . Sustained modification of a wildtype mosquito population using a homing based strategy, resulting in population suppression, has also been reported (26) . ...
Article
Full-text available
Significance Gene drive can spread beneficial traits through populations, but will never be a one-shot project in which one genetic element provides all desired modifications, for an indefinitely long time. Here, we show that gene drive-mediated population modification in Drosophila can be overwritten with new content while eliminating old, using Cleave and Rescue ( ClvR ) selfish genetic elements. The ability to carry out cycles of modification that create and then leave behind a modest genetic footprint while entering and exiting a population provides important points of control. It makes possible the replacement of broken elements, upgrades with new elements that better carry out their tasks, and/or provide new functions, all while promoting the removal of modifications no longer needed.
... CRISPR technology has greatly facilitated this type of engineering because CRISPR-Cas9 is a site-directed nuclease. The other class relies on biased offspring survival, many of which are known as 'killer-rescue' systems (Chen et al., 2007;Gould et al., 2008;Marshall & Hay, 2011;Legros et al., 2013;Akbari et al., 2013;Akbari et al., 2014;Buchman et al., 2018;Oberhofer, Ivy & Hay, 2019;Champer et al., 2019a). One of the major differences between these two classes of drive elements is the speed and ease with which they spread. ...
Article
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Gene drives may be used in two ways to curtail vectored diseases. Both involve engineering the drive to spread in the vector population. One approach uses the drive to directly depress vector numbers, possibly to extinction. The other approach leaves intact the vector population but suppresses the disease agent during its interaction with the vector. This second application may use a drive engineered to carry a genetic cargo that blocks the disease agent. An advantage of the second application is that it is far less likely to select vector resistance to block the drive, but the disease agent may instead evolve resistance to the inhibitory cargo. However, some gene drives are expected to spread so fast and attain such high coverage in the vector population that, if the disease agent can evolve resistance only gradually, disease eradication may be feasible. Here we use simple models to show that spatial structure in the vector population can greatly facilitate persistence and evolution of resistance by the disease agent. We suggest simple approaches to avoid some types of spatial structure, but others may be intrinsic to the populations being challenged and difficult to overcome.
... In Aedes aegypti, several regulatory elements able to drive gene expression in a tissue-and temporal-specific manner have been identified through extensive study (Akbari et al. 2013a) and transgenesis (Coates et al. 1999;Kokoza et al. 2000;Moreira et al. 2000;Smith et al. 2007). Future functional characterization of uncharacterized genes and regulatory elements may lead to the development of innovative genetic population control technologies such as precision guided sterile males (Kandul et al. 2019b), and gene drive systems (Akbari et al. 2013b(Akbari et al. , 2014aChamper et al. 2016;Buchman et al. 2018bBuchman et al. , 2018aKandul et al. 2019aKandul et al. , 2019bLi et al. 2019) which can be linked to anti-pathogen effectors (Buchman et al. , 2019b potentially providing paradigm-shifting technologies to control this worldwide human disease vector. Overall, our results provide a comprehensive snapshot of gene expression dynamics in the development of Ae. albopictus mosquitoes. ...
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Aedes albopictus mosquitoes are important vectors for a number of human pathogens including the Zika, dengue, and chikungunya viruses. Capable of displacing Aedes aegypti populations, it adapts to cooler environments which increases its geographical range and transmission potential. There are limited control strategies for Aedes albopictus mosquitoes which is likely attributed to the lack of comprehensive biological studies on this emerging vector. To fill this void, here using RNAseq we characterized Aedes albopictus mRNA expression profiles at 47 distinct time points throughout development providing the first high-resolution comprehensive view of the developmental transcriptome of this worldwide human disease vector. This enabled us to identify several patterns of shared gene expression among tissues as well as sex-specific expression patterns. Moreover, to illuminate the similarities and differences between Aedes aegypti, a related human disease vector, we performed a comparative analysis using the two developmental transcriptomes. We identify life stages were the two species exhibited significant differential expression among orthologs. These findings provide insights into the similarities and differences between Aedes albopictus and Aedes aegypti mosquito biology. In summary, the results generated from this study should form the basis for future investigations on the biology of Aedes albopictus mosquitoes and provide a goldmine resource for the development of transgene-based vector control strategies.
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Gene drives alleles that can bias their own inheritance are a promising way to engineer populations for control of disease vectors, invasive species, and agricultural pests. Recent advancements in the field have yielded successful examples of powerful suppression type drives and confined modification type drives, but developing confined suppression drives has proven more difficult. This is because the necessary power for strong suppression is often incompatible with the characteristics needed for drive confinement. However, one type of CRISPR toxin-antidote drive may be strong enough and confined, the TADE (Toxin-Antidote Dominant Embryo) suppression drive. By disrupting a haplolethal target gene and a haplosufficient female fertility gene, this drive quickly eliminates wild-type alleles and eventually induces population suppression. It has been shown to perform effectively in panmictic populations. However, confinement in spatial scenarios may be substantially different. Here, we use a reaction-diffusion model to assess the performance of TADE suppression drive in continuous space. We measure the drive wave advance speed while varying several performance parameters and find that moderate fitness costs or embryo cutting (from maternally deposited nuclease) can eliminate the ability of the drive to form a wave of advance. We assess the release size required for the drive to propagate, and finally, we investigate migration corridor scenarios. Depending on the corridor size and dispersal, it is often possible for the drive to suppress one population and then persist in the corridor without invading the second population. This prevents re-invasion by wild-type, which may be a particularly desirable outcome in some scenarios. Thus, even imperfect variants of TADE suppression drive may be excellent candidates for confined population suppression.
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With their ability to rapidly increase in frequency, gene drives can be used to modify or suppress target populations after an initial release of drive-containing individuals. Recent advances in this field have revealed many possibilities for different types of drives, and several of these have been realized in experimental demonstrations. These drives all have unique advantages and disadvantages related to their ease of construction, confinement, and capacity to act as a modification or suppression system. While many properties of these drives have been explored in modelling studies, assessment of these drives in continuous space environments has been limited, often focusing on outcomes rather than fundamental properties. Here, we conduct a comparative analysis of many different gene drive types that have the capacity to form a wave of advance against wild-type alleles in one-dimensional continuous space. We evaluate the drive wave speed as a function of drive performance and ecological parameters, which reveals substantial differences between drive performance in panmictic versus spatial environments. In particular, we find that suppression drive waves are uniquely vulnerable to fitness costs and undesired CRISPR cleavage activity that can form resistance alleles in embryos by maternal deposition. Some drives, though, retain robust characteristics even with widely varying performance characteristics. To gain a better understanding of drive waves, we compare panmictic performance of drives across the full range of drive frequencies. We find that rates of wild-type allele removal in panmictic setting is correlated with drive wave speed, though this is also affected by a range of other factors. Overall, our results provide a useful resource for understanding the performance of drives in continuous spatial environments, which may be most representative of potential drive deployment in many relevant scenarios.
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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.
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.
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.
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Speciation constrains the flow of genetic information between populations of sexually reproducing organisms. Gaining control over mechanisms of speciation would enable new strategies to manage wild populations of disease vectors, agricultural pests, and invasive species. Additionally, such control would provide safe biocontainment of transgenes and gene drives. Here, we demonstrate a general approach to create engineered genetic incompatibilities (EGIs) in the model insect Drosophila melanogaster. EGI couples a dominant lethal transgene with a recessive resistance allele. Strains homozygous for both elements are fertile and fecund when they mate with similarly engineered strains, but incompatible with wild-type strains that lack resistant alleles. EGI genotypes can also be tuned to cause hybrid lethality at different developmental life-stages. Further, we demonstrate that multiple orthogonal EGI strains of D. melanogaster can be engineered to be mutually incompatible with wild-type and with each other. EGI is a simple and robust approach in multiple sexually reproducing organisms.
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Robyn Raban and Omar Akbari describe a day in the life of the mosquito insectary team at the University of California, San Diego, outlining the procedures, goals, and types of systems they are engineering to control mosquito-transmitted diseases.
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Self-limiting gene drive allows control over the spread and fate of linked traits. Cleave and Rescue (ClvR) elements create self-sustaining drive and comprise a DNA sequence-modifying enzyme (Cas9-gRNAs, Cleaver) that disrupts an essential gene, and a tightly linked, uncleavable version of the essential gene (Rescue). ClvR spreads by creating conditions in which those without it die because they lack essential gene function. We show that when ClvR is implemented in a 2-locus format, with key elements - Rescue (and Cargo), and Cas9 and/or gRNAs - located at different genomic positions, spread of the Rescue is self-limiting. Drive strength and duration are determined by a recombination rate-dependent generational clock, providing an important point of control for different ecological and regulatory contexts. We implement 2-locus ClvR in Drosophila. Rescue spreads to high frequency in a Cas9-dependent manner, while the frequency of Cas9 decreases, demonstrating transient drive and loss of future drive potential.
Preprint
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Self-limiting gene drive allows control over the spread and fate of linked traits. Cleave and Rescue (ClvR) elements create self-sustaining drive and comprise a DNA sequence-modifying enzyme (Cas9-gRNAs, Cleaver) that disrupts an essential gene, and a tightly linked, uncleavable version of the essential gene (Rescue). ClvR spreads by creating conditions in which those without it die because they lack essential gene function. We show that when ClvR is implemented in a 2-locus format, with key elements - Rescue (and Cargo), and Cas9 and/or gRNAs - located at different genomic positions, spread of the Rescue is self-limiting. Drive strength and duration are determined by a recombination rate-dependent generational clock, providing an important point of control for different ecological and regulatory contexts. We implement 2-locus ClvR in Drosophila. Rescue spreads to high frequency in a Cas9-dependent manner, while the frequency of Cas9 decreases, demonstrating transient drive and loss of future drive potential.
Article
Vector-borne diseases, such as dengue, Zika and malaria, are a major cause of morbidity and mortality worldwide. These diseases have proven difficult to control and currently available management tools are insufficient to eliminate them in many regions. Gene drives have the potential to revolutionize vector-borne disease control. This suite of technologies has advanced rapidly in recent years as a result of the availability of new, more efficient gene editing technologies. Gene drives can favorably bias the inheritance of a linked disease-refractory gene, which could possibly be exploited (i) to generate a vector population incapable of transmitting disease or (ii) to disrupt an essential gene for viability or fertility, which could eventually eliminate a population. Importantly, gene drives vary in characteristics such as their transmission efficiency, confinability and reversibility, and their potential to develop resistance to the drive mechanism. Here, we discuss recent advancements in the gene drive field, and contrast the benefits and limitations of a variety of technologies, as well as approaches to overcome these limitations. We also discuss the current state of each gene drive technology and the technical considerations that need to be addressed on the pathway to field implementation. While there are still many obstacles to overcome, recent progress has brought us closer than ever before to genetic-based vector modification as a tool to support vector-borne disease elimination efforts worldwide.
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Homing based gene drives (HGD) possess the potential to spread linked cargo genes into natural populations and are poised to revolutionize population control of animals. Given that host encoded genes have been identified that are important for pathogen transmission, targeting these genes using guide RNAs as cargo genes linked to drives may provide a robust method to prevent disease transmission. However, effectiveness of the inclusion of additional guide RNAs that target separate genes has not been thoroughly explored. To test this approach, we generated a split-HGD in Drosophila melanogaster that encoded a drive linked effector consisting of a second gRNA engineered to target a separate host-encoded gene, which we term a gRNA-mediated effector (GME). This design enabled us to assess homing and knockout efficiencies of two target genes simultaneously, and also explore the timing and tissue specificity of Cas9 expression on cleavage/homing rates. We demonstrate that inclusion of a GME can result in high efficiency of disruption of both genes during super-Mendelian propagation of split-HGD. Furthermore, both genes were knocked out one generation earlier than expected indicating the robust somatic expression of Cas9 driven by Drosophila germline-limited promoters. We also assess the efficiency of 'shadow drive' generated by maternally deposited Cas9 protein and accumulation of drive-induced resistance alleles along multiple generations, and discuss design principles of HGD that could mitigate the accumulation of resistance alleles while incorporating a GME.
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1.Malaria, dengue, Zika, and other mosquito‐borne diseases continue to pose a major global health burden through much of the world, despite the widespread distribution of insecticide‐based tools and antimalarial drugs. The advent of CRISPR/Cas9‐based gene editing and its demonstrated ability to streamline the development of gene drive systems has reignited interest in the application of this technology to the control of mosquitoes and the diseases they transmit. The versatility of this technology has enabled a wide range of gene drive architectures to be realized, creating a need for their population‐level and spatial dynamics to be explored. 2.We present MGDrivE (Mosquito Gene Drive Explorer): a simulation framework designed to investigate the population dynamics of a variety of gene drive architectures and their spread through spatially‐explicit mosquito populations. A key strength of the MGDrivE framework is its modularity: a) a genetic inheritance module accommodates the dynamics of gene drive systems displaying userdefined inheritance patterns, b) a population dynamic module accommodates the life history of a variety of mosquito disease vectors and insect agricultural pests, and c) a landscape module generates the metapopulation model by which insect populations are connected via migration over space. 3.Example MGDrivE simulations are presented to demonstrate the application of the framework to CRISPR/Cas9‐based homing gene drive for: a) driving a disease‐refractory gene into a population (i.e. population replacement), and b) disrupting a gene required for female fertility (i.e. population suppression), incorporating homing‐resistant alleles in both cases. Further documentation and use examples are provided at the project's Github repository. 4.MGDrivE is an open‐source R package freely available on CRAN. We intend the package to provide a flexible tool capable of modeling novel inheritance‐modifying constructs as they are proposed and become available. The field of gene drive is moving very quickly, and we welcome suggestions for future development.
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Synthetic gene drive systems possess enormous potential to replace, alter, or suppress wild populations of significant disease vectors and crop pests; however, their utility in diverse populations remains to be demonstrated. Here, we report the creation of the first-ever synthetic Medea gene drive element in a major worldwide crop pest, D. suzukii . We demonstrate that this drive element, based on an engineered maternal “toxin” coupled with a linked embryonic “antidote,” is capable of biasing Mendelian inheritance rates with up to 100% efficiency. However, we find that drive resistance, resulting from naturally occurring genetic variation and associated fitness costs, can hinder the spread of such an element. Despite this, our results suggest that this element could maintain itself at high frequencies in a wild population, and spread to fixation, if either its fitness costs or toxin resistance were reduced, providing a clear path forward for developing future such systems.
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Approximately two years ago, two of us (E.B. and V.G.) demonstrated the first experimental application of CRISPR–Cas9 to 'drive' a desired trait throughout a population of fruit flies. In November 2015, this same team at the University of California, San Diego, joined with A.A.J. and others at the University of California, Irvine, to develop a CRISPR-based gene drive for population modification of the malaria vector mosquito Anopheles stephensi. A month later, a group in the United Kingdom applied a CRISPR-based gene drive to another malaria vector, Anopheles gambiae.
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Aedes aegypti, the primary vector of dengue, yellow fever and Zika flaviviruses, consists of at least two subspecies. Aedes aegypti (Aaa) is light in color, has pale scales on the first abdominal tergite, oviposits in artificial containers, and preferentially feeds on humans. Aedes aegypti formosus (Aaf), has a dark cuticle, is restricted to sub-Saharan Africa, has no pale scales on the first abdominal tergite and frequently oviposits in natural containers. Scale patterns correlate with cuticle color in East Africa but not in Senegal, West Africa where black cuticle mosquitoes display a continuum of scaling patterns and breed domestically indoors. An earlier laboratory study did not indicate any pre- or postzygotic barriers to gene flow between Aaa and Aaf in East Africa. However, similar attempts to construct F1 intercross families between Aaa laboratory strains and Senegal Ae. aegypti (SenAae) failed due to poor F1 oviposition and low F2 egg-to-adult survival. Insemination and assortative mating experiments failed to identify prezygotic mating barriers. Backcrosses were performed to test for postzygotic isolation patterns consistent with Haldane's rule modified for species, like Aedes, that have an autosomal sex determining locus (SDL). Egg-pupal survival was predicted to be low in females mated to hybrid F1 males but average when a male mates with a hybrid F1 female. Survival was in fact significantly reduced when females mated to hybrid males but egg-pupal survival was significantly increased when males were mated to hybrid F1 females. These observations are therefore inconclusive with regards to Haldane's rule. Basic cytogenetic analyses and Fluorescent In Situ Hybridization (FISH) experiments were performed to compare SenAae strains with the IB12 strain of Aaa that was used for genome sequencing and physical mapping. Some SenAae strains had longer chromosomes than IB12 and significantly different centromeric indices on chromosomes 1 and 3. DAPI staining was used to identify AT-rich regions, chromomycin A3 following pretreatment with barium hydroxide stained for GC-rich regions and stained the ribosomal RNA locus and YOYO-1 was used to test for differential staining. Chromosome patterns in SenAae strains revealed by these three stains differed from those in IB12. For FISH, 40 BAC clones previously physically mapped on Aaa chromosomes were used to test for chromosome rearrangements in SenAae relative to IB12. Differences in the order of markers identified two chromosomal rearrangements between IB12 and SenAae strains. The first rearrangement involves two overlapping pericentric (containing the centromere) inversions in chromosome 3 or an insertion of a large fragment into the 3q arm. The second rearrangement is close to the centromere on the p arm of chromosome 2. Linkage analysis of the SDL and the white-eye locus identified a likely chromosomal rearrangement on chromosome 1. The reproductive incompatibility observed within SenAae and between SenAae and Aaa may be generally associated with chromosome rearrangements on all three chromosomes and specifically caused by pericentric inversions on chromosomes 2 and 3.
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Chromosomal translocation is the most common form of chromosomal abnormality and is often associated with congenital genetic disorders, infertility, and cancers. The lack of cellular and animal models for chromosomal translocations, however, has hampered our ability to understand the underlying disease mechanisms and to develop new therapies. Here, we show that site-specific chromosomal translocations can be generated in mouse embryonic stem cells (mESCs) via CRISPR/Cas9. Mouse ESCs carrying translocated chromosomes can be isolated and expanded to establish stable cell lines. Furthermore, chimeric mice can be generated by injecting these mESCs into host blastocysts. The establishment of ESC-based cellular and animal models of chromosomal translocation by CRISPR/Cas9 provides a powerful platform for understanding the effect of chromosomal translocation and for the development of new therapeutic strategies.
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Many genes play essential roles in development and fertility; their disruption leads to growth arrest or sterility. Genetic balancers have been widely used to study essential genes in many organisms. However, it is technically challenging and laborious to generate and maintain the loss of function mutations of essential genes. The CRISPR/Cas9 technology has been successfully applied for gene editing and chromosome engineering. Here, we developed a method to induce chromosomal translocations and produce genetic balancers using the CRISPR/Cas9 technology, and applied this approach to edit essential genes in Caenorhabditis elegans. The co-injection of dual sgRNAs targeting genes on different chromosomes resulted in reciprocal translocation between non-homologous chromosomes. These animals with chromosomal translocations were subsequently crossed with animals that contain normal sets of chromosomes. The F1 progeny were subjected to a second round of Cas9-mediated gene editing. Through this method, we successfully produced nematode strains with specified chromosomal translocations and generated a number of loss of function alleles of two essential genes (csr-1 and mes-6). Therefore, our method provides an easy and efficient approach to generate and maintain loss of function alleles of essential genes with detailed genetic background information.
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Gene drive systems promote the spread of genetic elements through populations by assuring they are inherited more often than Mendelian segregation would predict (see the figure). Natural examples of gene drive from Drosophila include sex-ratio meiotic drive, segregation distortion, and replicative transposition. Synthetic drive systems based on selective embryonic lethality or homing endonucleases have been described previously in Drosophila melanogaster (1–3), but they are difficult to build or are limited to transgenic populations. In contrast, RNAguided gene drives based on the CRISPR/Cas9 nuclease can, in principle, be constructed by any laboratory capable of making transgenic organisms (4). They have tremendous potential to address global problems in health, agriculture, and conservation, but their capacity to alter wild populations outside the laboratory demands caution (4–7). Just as researchers working with self-propagating pathogens must ensure that these agents do not escape to the outside world, scientists working in the laboratory with gene drive constructs are responsible for keeping them confined (4, 6, 7).
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The increasing burden of dengue, and the relative failure of traditional vector control programs highlight the need to develop new control methods. SIT using self-limiting genetic technology is one such promising method. A self-limiting strain of Aedes aegypti, OX513A, has already reached the stage of field evaluation. Sustained releases of OX513A Ae. aegypti males led to 80% suppression of a target wild Ae. aegypti population in the Cayman Islands in 2010. Here we describe sustained series of field releases of OX513A Ae. aegypti males in a suburb of Juazeiro, Bahia, Brazil. This study spanned over a year and reduced the local Ae. aegypti population by 95% (95% CI: 92.2%-97.5%) based on adult trap data and 81% (95% CI: 74.9-85.2%) based on ovitrap indices compared to the adjacent no-release control area. The mating competitiveness of the released males (0.031; 95% CI: 0.025-0.036) was similar to that estimated in the Cayman trials (0.059; 95% CI: 0.011 - 0.210), indicating that environmental and target-strain differences had little impact on the mating success of the OX513A males. We conclude that sustained release of OX513A males may be an effective and widely useful method for suppression of the key dengue vector Ae. aegypti. The observed level of suppression would likely be sufficient to prevent dengue epidemics in the locality tested and other areas with similar or lower transmission.
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Introgressive hybridization is now recognized as a widespread phenomenon, but its role in evolution remains contested. Here we use newly available reference genome assemblies to investigate phylogenetic relationships and introgression in a medically important group of Afrotropical mosquito sibling species. We have identified the correct species branching order to resolve a contentious phylogeny, and show that lineages leading to the principal vectors of human malaria were among the first to split. Pervasive autosomal introgression between these malaria vectors means that only a small fraction of the genome, mainly on the X chromosome, has not crossed species boundaries. Our results suggest that traits enhancing vectorial capacity may be gained through interspecific gene flow, including between non-sister species. Copyright © 2014, American Association for the Advancement of Science.
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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The effects that coding region single-nucleotide polymorphisms or mutations have on gene expression have been well documented, predominantly owing to their association with disease. The effects of structural chromosomal rearrangements are also receiving increasing attention with the development of new techniques that allow accurate, high-resolution data, whether genomic interaction or transcriptome data, to be generated right down to the single-cell level. Over the past 18 months, these advances in experimental techniques have been used to further confirm and delineate the substantial effects that chromosome rearrangements can have on the regulation of gene expression and provide evidence of direct links between the two.
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Cancer-related human chromosomal translocations are generated through the illegitimate joining of two non-homologous chromosomes affected by double-strand breaks (DSB). Effective methodologies to reproduce precise reciprocal tumour-associated chromosomal translocations are required to gain insight into the initiation of leukaemia and sarcomas. Here we present a strategy for generating cancer-related human chromosomal translocations in vitro based on the ability of the RNA-guided CRISPR-Cas9 system to induce DSBs at defined positions. Using this approach we generate human cell lines and primary cells bearing chromosomal translocations resembling those described in acute myeloid leukaemia and Ewing's sarcoma at high frequencies. FISH and molecular analysis at the mRNA and protein levels of the fusion genes involved in these engineered cells reveal the reliability and accuracy of the CRISPR-Cas9 approach, providing a powerful tool for cancer studies.
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Background: Transgenic constructs intended to be stably established at high frequencies in wild populations have been demonstrated to "drive" from low frequencies in experimental insect populations. Linking such population transformation constructs to genes which render them unable to transmit pathogens could eventually be used to stop the spread of vector-borne diseases like malaria and dengue. Results: Generally, population transformation constructs with only a single transgenic drive mechanism have been envisioned. Using a theoretical modelling approach we describe the predicted properties of a construct combining autosomal Medea and underdominant population transformation systems. We show that when combined they can exhibit synergistic properties which in broad circumstances surpass those of the single systems. Conclusion: With combined systems, intentional population transformation and its reversal can be achieved readily. Combined constructs also enhance the capacity to geographically restrict transgenic constructs to targeted populations. It is anticipated that these properties are likely to be of particular value in attracting regulatory approval and public acceptance of this novel technology.
Article
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The idea of introducing genetic modifications into wild populations of insects to stop them from spreading diseases is more than 40 years old. Synthetic disease refractory genes have been successfully generated for mosquito vectors of dengue fever and human malaria. Equally important is the development of population transformation systems to drive and maintain disease refractory genes at high frequency in populations. We demonstrate an underdominant population transformation system in Drosophila melanogaster that has the property of being both spatially self-limiting and reversible to the original genetic state. Both population transformation and its reversal can be largely achieved within as few as 5 generations. The described genetic construct {Ud} is composed of two genes; (1) a UAS-RpL14.dsRNA targeting RNAi to a haploinsufficient gene RpL14 and (2) an RNAi insensitive RpL14 rescue. In this proof-of-principle system the UAS-RpL14.dsRNA knock-down gene is placed under the control of an Actin5c-GAL4 driver located on a different chromosome to the {Ud} insert. This configuration would not be effective in wild populations without incorporating the Actin5c-GAL4 driver as part of the {Ud} construct (or replacing the UAS promoter with an appropriate direct promoter). It is however anticipated that the approach that underlies this underdominant system could potentially be applied to a number of species.
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Mosquito-borne diseases are causing a substantial burden of mortality, morbidity and economic loss in many parts of the world, despite current control efforts, and new complementary approaches to controlling these diseases are needed. One promising class of new interventions under development involves the heritable modification of the mosquito by insertion of novel genes into the nucleus or of Wolbachia endosymbionts into the cytoplasm. Once released into a target population, these modifications can act to reduce one or more components of the mosquito population's vectorial capacity (e.g. the number of female mosquitoes, their longevity or their ability to support development and transmission of the pathogen). Some of the modifications under development are designed to be self-limiting, in that they will tend to disappear over time in the absence of recurrent releases (and hence are similar to the sterile insect technique, SIT), whereas other modifications are designed to be self-sustaining, spreading through populations even after releases stop (and hence are similar to traditional biological control). Several successful field trials have now been performed with Aedes mosquitoes, and such trials are helping to define the appropriate developmental pathway for this new class of intervention.
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Significance Populations of the African malaria vector, Anopheles gambiae , are structured into M and S forms. All current work assumes the two rarely hybridize. Here we show this assumption is false. We demonstrate ( i ) significant exchange of genes between the two forms, even though ( ii ) hybrids have reduced fitness and ( iii ) the gene exchange process is spatially and temporally dynamic. For malaria, it is important to determine if genes for traits like insecticide resistance are shared between forms. For evolutionary biologists, this work confirms that this mosquito is a good model for studying if and how species may evolve in cases where there is ongoing gene flow.
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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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