73 results found
Pescod P, Bevivino G, Anthousi A, et al., 2023, Measuring the Impact of Genetic Heterogeneity and Chromosomal Inversions on the Efficacy of CRISPR-Cas9 Gene Drives in Different Strains of Anopheles gambiae., CRISPR J
The human malaria vector Anopheles gambiae is becoming increasingly resistant to insecticides, spurring the development of genetic control strategies. CRISPR-Cas9 gene drives can modify a population by creating double-stranded breaks at highly specific targets, triggering copying of the gene drive into the cut site ("homing"), ensuring its inheritance. The DNA repair mechanism responsible requires homology between the donor and recipient chromosomes, presenting challenges for the invasion of laboratory-developed gene drives into wild populations of target species An. gambiae species complex, which show high levels of genome variation. Two gene drives (vas2-5958 and zpg-7280) were introduced into three An. gambiae strains collected across Africa with 5.3-6.6% variation around the target sites, and the effect of this variation on homing was measured. Gene drive homing across different karyotypes of the 2La chromosomal inversion was also assessed. No decrease in gene drive homing was seen despite target site heterology, demonstrating the applicability of gene drives to wild populations.
Page N, Taxiarchi C, Tonge D, et al., 2023, Single-cell profiling of Anopheles gambiae spermatogenesis defines the onset of meiotic silencing and premeiotic overexpression of the X chromosome., Commun Biol, Vol: 6
Understanding development and genetic regulation in the Anopheles gambiae germline is essential to engineer effective genetic control strategies targeting this malaria mosquito vector. These include targeting the germline to induce sterility or using regulatory sequences to drive transgene expression for applications such as gene drive. However, only very few germline-specific regulatory elements have been characterised with the majority showing leaky expression. This has been shown to considerably reduce the efficiency of current genetic control strategies, which rely on regulatory elements with more tightly restricted spatial and/or temporal expression. Meiotic silencing of the sex chromosomes limits the flexibility of transgene expression to develop effective sex-linked genetic control strategies. Here, we build on our previous study, dissecting gametogenesis into four distinct cell populations, using single-cell RNA sequencing to define eight distinct cell clusters and associated germline cell-types using available marker genes. We reveal overexpression of X-linked genes in a distinct cluster of pre-meiotic cells and document the onset of meiotic silencing of the X chromosome in a subcluster of cells in the latter stages of spermatogenesis. This study provides a comprehensive dataset, characterising the expression of distinct cell types through spermatogenesis and widening the toolkit for genetic control of malaria mosquitoes.
Morianou I, Crisanti A, Nolan T, et al., 2022, CRISPR-Mediated Cassette Exchange (CriMCE): A Method to Introduce and Isolate Precise Marker-Less Edits, CRISPR JOURNAL, Vol: 5, Pages: 868-876, ISSN: 2573-1599
Fuchs S, Garrood WT, Beber A, et al., 2021, Resistance to a CRISPR-based gene drive at an evolutionarily conserved site is revealed by mimicking genotype fixation, PLoS Genetics, Vol: 17, Pages: 1-19, ISSN: 1553-7390
CRISPR-based homing gene drives can be designed to disrupt essential genes whilst biasing their own inheritance, leading to suppression of mosquito populations in the laboratory. This class of gene drives relies on CRISPR-Cas9 cleavage of a target sequence and copying (‘homing’) therein of the gene drive element from the homologous chromosome. However, target site mutations that are resistant to cleavage yet maintain the function of the essential gene are expected to be strongly selected for. Targeting functionally constrained regions where mutations are not easily tolerated should lower the probability of resistance. Evolutionary conservation at the sequence level is often a reliable indicator of functional constraint, though the actual level of underlying constraint between one conserved sequence and another can vary widely. Here we generated a novel adult lethal gene drive (ALGD) in the malaria vector Anopheles gambiae, targeting an ultra-conserved target site in a haplosufficient essential gene (AGAP029113) required during mosquito development, which fulfils many of the criteria for the target of a population suppression gene drive. We then designed a selection regime to experimentally assess the likelihood of generation and subsequent selection of gene drive resistant mutations at its target site. We simulated, in a caged population, a scenario where the gene drive was approaching fixation, where selection for resistance is expected to be strongest. Continuous sampling of the target locus revealed that a single, restorative, in-frame nucleotide substitution was selected. Our findings show that ultra-conservation alone need not be predictive of a site that is refractory to target site resistance. Our strategy to evaluate resistance in vivo could help to validate candidate gene drive targets for their resilience to resistance and help to improve predictions of the invasion dynamics of gene drives in field populations.
Hammond A, Pollegioni P, Persampieri T, et al., 2021, Gene-drive suppression of mosquito populations in large cages as a bridge between lab and field., Nature Communications, Vol: 12, Pages: 1-9, ISSN: 2041-1723
CRISPR-based gene-drives targeting the gene doublesex in the malaria vector Anopheles gambiae effectively suppressed the reproductive capability of mosquito populations reared in small laboratory cages. To bridge the gap between laboratory and the field, this gene-drive technology must be challenged with vector ecology.Here we report the suppressive activity of the gene-drive in age-structured An. gambiae populations in large indoor cages that permit complex feeding and reproductive behaviours.The gene-drive element spreads rapidly through the populations, fully supresses the population within one year and without selecting for resistance to the gene drive. Approximate Bayesian computation allowed retrospective inference of life-history parameters from the large cages and a more accurate prediction of gene-drive behaviour under more ecologically-relevant settings.Generating data to bridge laboratory and field studies for invasive technologies is challenging. Our study represents a paradigm for the stepwise and sound development of vector control tools based on gene-drive.
Grigoraki L, Cowlishaw R, Nolan T, et al., 2021, CRISPR/Cas9 modified An. gambiae carrying kdr mutation L1014F functionally validate its contribution in insecticide resistance and combined effect with metabolic enzymes, PLOS GENETICS, Vol: 17, ISSN: 1553-7404
Quinn C, Anthousi A, Wondji C, et al., 2021, CRISPR-mediated knock-in of transgenes into the malaria vector Anopheles funestus, G3-GENES GENOMES GENETICS, Vol: 11, ISSN: 2160-1836
Grigoraki L, Cowlishaw R, Nolan T, et al., 2021, CRISPR/Cas9 modified <i>An. gambiae</i> carrying kdr mutation L1014F functionally validate its contribution in insecticide resistance and interaction with metabolic enzymes
<jats:title>Abstract</jats:title><jats:p>Insecticide resistance in <jats:italic>Anopheles</jats:italic> mosquitoes is a major obstacle in maintaining the momentum in reducing the malaria burden; mitigating strategies require improved understanding of the underlying mechanisms. Mutations in the target site of insecticides (the voltage gated sodium channel for the most widely used pyrethroid class) and over-expression of detoxification enzymes are commonly reported, but their relative contribution to phenotypic resistance remain poorly understood. Here we present a genome editing pipeline to introduce single nucleotide polymorphisms in <jats:italic>An. gambiae</jats:italic> which we have used to study the effect of the classical kdr mutation L1014F (L995F based on <jats:italic>An. gambiae</jats:italic> numbering), one of the most widely distributed resistance alleles. Introduction of 1014F in an otherwise fully susceptible genetic background increased levels of resistance to all tested pyrethroids and DDT ranging from 9.9-fold for permethrin to >24-fold for DDT. The introduction of the 1014F allele was sufficient to reduce mortality of mosquitoes after exposure to deltamethrin treated bednets, even as the only resistance mechanism present. When 1014F was combined with over-expression of glutathione transferase Gste2, resistance to permethrin increased further demonstrating the critical combined effect between target site resistance and detoxification enzymes <jats:italic>in vivo</jats:italic>. We also show that mosquitoes carrying the 1014F allele in homozygosity showed fitness disadvantages including increased mortality at the larval stage and a reduction in fecundity and adult longevity, which can have consequences for the strength of selection that will apply to this allele in the field.</jats:p><jats:sec><jats:title>Author Summary</jats:title><jats:p>Escalation o
Hammond A, Pollegioni P, Persampieri T, et al., 2021, Population suppression of the malaria vector Anopheles gambiae by gene drive technology: A large-cage indoor study bridging the gap between laboratory and field testing
<jats:title>Abstract</jats:title> <jats:p>CRISPR-based gene drives are self-sustaining genetic elements that have been recently generated in the laboratory with the aim to develop potent genetic vector control measures targeting disease vectors including Anopheles gambiae. We have shown that a gene drive directed against the gene doublesex (dsx) effectively suppressed the reproductive capability of mosquito populations reared in small laboratory cages. These experiments, though informative, do not recapitulate the complexity of mosquito behaviour in natural environments. Additional information is needed to bridge the gap between laboratory and the field to validate the vector control potential of the technology. We have investigated the suppressing activity of dsx gene drive strain Ag(QFS)1 on age-structured populations of Anopheles gambiae in large indoor cages that provide a more challenging ecology by more closely mimicking natural conditions and stimulating complex mosquito behaviours. Under these conditions, the Ag(QFS)1 drive spreads rapidly from a single release to the indoor large-cage populations at low initial frequency, leading to full population suppression within one year and without inducing resistance to the gene drive. Initial stochastic simulations of the expected population dynamics, as based on life history parameters estimated in small cages, did not fully capture the observed dynamics in the large cages. Thus, we used the method of approximate Bayesian computation to better estimate population dynamics in the more realistic ecological setting in large cages, allowing the mosquitoes to show a complex feeding and reproductive behaviour.Together, these results establish a new paradigm for generating data to bridge laboratory and field studies, and form an essential component in the stepwise and sound development of gene drive based vector control tools.</jats:p>
Quinn C, Anthousi A, Wondji C, et al., 2021, CRISPR-mediated knock-in of transgenes into the malaria vector<i>Anopheles funestus</i>
<jats:title>ABSTRACT</jats:title><jats:p>The ability to introduce mutations, or transgenes, of choice to precise genomic locations has revolutionised our ability to understand how genes and organisms work.</jats:p><jats:p>In many mosquito species that are vectors of various human disease, the advent of CRISPR genome editing tools has shed light on basic aspects of their biology that are relevant to their efficiency as disease vectors. This allows a better understanding of how current control tools work and opens up the possibility of novel genetic control approaches, such as gene drives, that deliberately introduce genetic traits into populations. Yet for the<jats:italic>Anopheles funestus</jats:italic>mosquito, a significant vector of malaria in sub-Saharan Africa and indeed the dominant vector species in many countries, transgenesis has yet to be achieved.</jats:p><jats:p>We describe herein an optimised transformation system based on the germline delivery of CRISPR components that allows efficient cleavage of a previously validated genomic site and preferential repair of these cut sites via homology-directed repair (HDR), which allows introduction of exogenous template sequence, rather than end-joining repair. The rates of transformation achieved are sufficiently high that it should be able to introduce alleles of choice to a target locus, and recover these, without the need to include additional dominant marker genes. Moreover, the high rates of HDR observed suggest that gene drives, which employ an HDR-type mechanism to ensure their proliferation in the genome, may be well suited to work in<jats:italic>An. funestus</jats:italic>.</jats:p>
O'Loughlin SM, Forster AJ, Fuchs S, et al., 2021, Ultra-conserved sequences in the genomes of highly diverse Anopheles mosquitoes, with implications for malaria vector control, G3-GENES GENOMES GENETICS, Vol: 11, ISSN: 2160-1836
Ma Q, Srivastav SP, Gamez S, et al., 2021, A mosquito small RNA genomics resource reveals dynamic evolution and host responses to viruses and transposons, GENOME RESEARCH, Vol: 31, Pages: 512-528, ISSN: 1088-9051
Nolan T, 2021, Control of malaria-transmitting mosquitoes using gene drives, PHILOSOPHICAL TRANSACTIONS OF THE ROYAL SOCIETY B-BIOLOGICAL SCIENCES, Vol: 376, ISSN: 0962-8436
Nolan T, 2021, Control of malaria-transmitting mosquitoes using gene drives., Philos Trans R Soc Lond B Biol Sci, Vol: 376
Gene drives are selfish genetic elements that can be re-designed to invade a population and they hold tremendous potential for the control of mosquitoes that transmit disease. Much progress has been made recently in demonstrating proof of principle for gene drives able to suppress populations of malarial mosquitoes, or to make them refractory to the Plasmodium parasites they transmit. This has been achieved using CRISPR-based gene drives. In this article, I will discuss the relative merits of this type of gene drive, as well as barriers to its technical development and to its deployment in the field as malaria control. This article is part of the theme issue 'Novel control strategies for mosquito-borne diseases'.
Kranjc N, Crisanti A, Nolan T, et al., 2021, Anopheles gambiae genome conservation as a resource for rational gene drive target site selection., Insects, Vol: 12, Pages: 1-12, ISSN: 2075-4450
The increase in molecular tools for the genetic engineering of insect pests and disease vectors, such as Anopheles mosquitoes that transmit malaria, has led to an unprecedented investigation of the genomic landscape of these organisms. The understanding of genome variability in wild mosquito populations is of primary importance for vector control strategies. This is particularly the case for gene drive systems, which look to introduce genetic traits into a population by targeting specific genomic regions. Gene drive targets with functional or structural constraints are highly desirable as they are less likely to tolerate mutations that prevent targeting by the gene drive and consequent failure of the technology. In this study we describe a bioinformatic pipeline that allows the analysis of whole genome data for the identification of highly conserved regions that can point at potential functional or structural constraints. The analysis was conducted across the genomes of 22 insect species separated by more than hundred million years of evolution and includes the observed genomic variation within field caught samples of Anopheles gambiae and Anopheles coluzzii, the two most dominant malaria vectors. This study offers insight into the level of conservation at a genome-wide scale as well as at per base-pair resolution. The results of this analysis are gathered in a data storage system that allows for flexible extraction and bioinformatic manipulation. Furthermore, it represents a valuable resource that could provide insight into population structure and dynamics of the species in the complex and benefit the development and implementation of genetic strategies to tackle malaria.
Hammond A, Karlsson X, Morianou I, et al., 2021, Regulating the expression of gene drives is key to increasing their invasive potential and the mitigation of resistance, PLOS GENETICS, Vol: 17, ISSN: 1553-7404
Simoni A, Hammond AM, Beaghton AK, et al., 2020, A male-biased sex-distorter gene drive for the human malaria vector Anopheles gambiae, Nature Biotechnology, Vol: 38, Pages: 1054-1060, ISSN: 1087-0156
Only female insects transmit diseases such as malaria, dengue and Zika; therefore, control methods that bias the sex ratio of insect offspring have long been sought. Genetic elements such as sex-chromosome drives can distort sex ratios to produce unisex populations that eventually collapse, but the underlying molecular mechanisms are unknown. We report a male-biased sex-distorter gene drive (SDGD) in the human malaria vector Anopheles gambiae. We induced super-Mendelian inheritance of the X-chromosome-shredding I-PpoI nuclease by coupling this to a CRISPR-based gene drive inserted into a conserved sequence of the doublesex (dsx) gene. In modeling of invasion dynamics, SDGD was predicted to have a quicker impact on female mosquito populations than previously developed gene drives targeting female fertility. The SDGD at the dsx locus led to a male-only population from a 2.5% starting allelic frequency in 10-14 generations, with population collapse and no selection for resistance. Our results support the use of SDGD for malaria vector control.
Gomulski LM, Manni M, Carraretto D, et al., 2020, Transcriptional variation of sensory-related genes in natural populations of Aedes albopictus, BMC Genomics, Vol: 21, ISSN: 1471-2164
BACKGROUND: The Asian tiger mosquito, Aedes albopictus, is a highly dangerous invasive vector of numerous medically important arboviruses including dengue, chikungunya and Zika. In four decades it has spread from tropical Southeast Asia to many parts of the world in both tropical and temperate climes. The rapid invasion process of this mosquito is supported by its high ecological and genetic plasticity across different life history traits. Our aim was to investigate whether wild populations, both native and adventive, also display transcriptional genetic variability for functions that may impact their biology, behaviour and ability to transmit arboviruses, such as sensory perception. RESULTS: Antennal transcriptome data were derived from mosquitoes from a native population from Ban Rai, Thailand and from three adventive Mediterranean populations: Athens, Greece and Arco and Trento from Italy. Clear inter-population differential transcriptional activity was observed in different gene categories related to sound perception, olfaction and viral infection. The greatest differences were detected between the native Thai and the Mediterranean populations. The two Italian populations were the most similar. Nearly one million quality filtered SNP loci were identified. CONCLUSION: The ability to express this great inter-population transcriptional variability highlights, at the functional level, the remarkable genetic flexibility of this mosquito species. We can hypothesize that the differential expression of genes, including those involved in sensory perception, in different populations may enable Ae. albopictus to exploit different environments and hosts, thus contributing to its status as a global vector of arboviruses of public health importance. The large number of SNP loci present in these transcripts represents a useful addition to the arsenal of high-resolution molecular markers and a resource that can be used to detect selective pressure and adaptive changes that may
Pollegioni P, North AR, Persampieri T, et al., 2020, Detecting the population dynamics of an autosomal sex ratio distorter transgene in malariavector mosquitoes, JOURNAL OF APPLIED ECOLOGY, Vol: 57, Pages: 2086-2096, ISSN: 0021-8901
Quinn CM, Nolan T, 2020, Nuclease -based gene drives, an innovative tool for insect vector control: advantages and challenges of the technology, CURRENT OPINION IN INSECT SCIENCE, Vol: 39, Pages: 77-83, ISSN: 2214-5745
James SL, Marshall JM, Christophides GK, et al., 2020, Toward the definition of efficacy and safety criteria for advancing gene drive-modified mosquitoes to field testing, Vector-Borne and Zoonotic Diseases, Vol: 20, Pages: 237-251, ISSN: 1530-3667
Mosquitoes containing gene drive systems are being developed as complementary tools to prevent transmission of malaria and other mosquito-borne diseases. As with any new tool, decision makers and other stakeholders will need to balance risks (safety) and benefits (efficacy) when considering the rationale for testing and deploying gene drive-modified mosquito products. Developers will benefit from standards for judging whether an investigational gene drive product meets acceptability criteria for advancing to field trials. Such standards may be formalized as preferred product characteristics and target product profiles, which describe the desired attributes of the product category and of a particular product, respectively. This report summarizes discussions from two scientific workshops aimed at identifying efficacy and safety characteristics that must be minimally met for an investigational gene drive-modified mosquito product to be deemed viable to move from contained testing to field release and the data that will be needed to support an application for first field release.
Beaghton AK, Hammond A, Nolan T, et al., 2019, Gene drive for population genetic control: non-functional resistance and parental effects, Proceedings of the Royal Society B: Biological Sciences, Vol: 286, Pages: 1-8, ISSN: 0962-8452
Gene drive is a natural process of biased inheritance that, in principle, could be used to control pest and vector populations. As with any form of pest control, attention should be paid to the possibility of resistance evolving. For nuclease-based gene drive aimed at suppressing a population, resistance could arise by changes in the target sequence that maintain function, and various strategies have been proposed to reduce the likelihood that such alleles arise. Even if these strategies are successful, it is almost inevitable that alleles will arise at the target site that are resistant to the drive but do not restore function, and the impact of such sequences on the dynamics of control has been little studied. We use population genetic modelling of a strategy targeting a female fertility gene to demonstrate that such alleles may be expected to accumulate, and thereby reduce the reproductive load on the population, if nuclease expression per se causes substantial heterozygote fitness effects or if parental (especially paternal) deposition of nuclease either reduces offspring fitness or affects the genotype of their germline. All these phenomena have been observed in synthetic drive constructs. It will, therefore, be important to allow for non-functional resistance alleles in predicting the dynamics of constructs in cage populations and the impacts of any field release.
Taxiarchi C, Kranjc N, Kriezis A, et al., 2019, High-resolution transcriptional profiling of Anopheles gambiae spermatogenesis reveals mechanisms of sex chromosome regulation, SCIENTIFIC REPORTS, Vol: 9, ISSN: 2045-2322
Bernardini F, Kriezis A, Galizi R, et al., 2019, Introgression of a synthetic sex ratio distortion system from Anopheles gambiae into Anopheles arabiensis (vol 9, 5158, 2019), SCIENTIFIC REPORTS, Vol: 9, ISSN: 2045-2322
Bernardini F, Kriezis A, Galizi R, et al., 2019, Introgression of a synthetic sex ratio distortion system from Anopheles gambiae into Anopheles arabiensis, Scientific Reports, Vol: 9, ISSN: 2045-2322
I-PpoI is a homing endonuclease that has a high cleavage activity and specificity for a conserved sequence within the ribosomal rDNA repeats, located in a single cluster on the Anopheles gambiae X chromosome. This property has been exploited to develop a synthetic sex ratio distortion system in this mosquito species. When I-PpoI is expressed from a transgene during spermatogenesis in mosquitoes, the paternal X chromosome is shredded and only Y chromosome-bearing sperm are viable, resulting in a male-biased sex ratio of >95% in the progeny. These distorter male mosquitoes can efficiently suppress caged wild-type populations, providing a powerful tool for vector control strategies. Given that malaria mosquito vectors belong to a species complex comprising at least two major vectors, we investigated whether the sex distorter I-PpoI, originally integrated in the A. gambiae genome, could be transferred via introgression to the sibling vector species Anopheles arabiensis. In compliance with Haldane’s rule, F1 hybrid male sterility is known to occur in all intercrosses among members of the Anopheles gambiae complex. A scheme based on genetic crosses and transgene selection was used to bypass F1 hybrid male sterility and introgress the sex distorter I-PpoI into the A. arabiensis genetic background. Our data suggest that this sex distortion technique can be successfully applied to target A. arabiensis mosquitoes.
Bernardini F, Haghighat-Khah RE, Galizi R, et al., 2018, Molecular tools and genetic markers for the generation of transgenic sexing strains in Anopheline mosquitoes, Parasites & Vectors, Vol: 11, ISSN: 1756-3305
Malaria is a serious global health burden, affecting more than 200 million people each year in over 90 countries, predominantly in Africa, Asia and the Americas. Since the year 2000, a concerted effort to combat malaria has reduced its incidence by more than 40%, primarily due to the use of insecticide-treated bednets, indoor residual spraying and artemisinin-based combination drug therapies. Nevertheless, the cost of control is expected to nearly triple over the next decade and the current downward trend in disease transmission is threatened by the rise of resistance to drugs and insecticides. Novel strategies that are sustainable and cost-effective are needed to help usher in an era of malaria elimination. The most effective strategies thus far have focussed on control of the mosquito vector. The sterile insect technique (SIT) is a potentially powerful strategy that aims to suppress mosquito populations through the unproductive mating of wild female mosquitoes with sterile males that are released en masse. The technique and its derivatives are currently not appropriate for malaria control because it is difficult to sterilise males without compromising their ability to mate, and because anopheline males cannot be easily separated from females, which if released, could contribute to disease transmission. Advances in genome sequencing technologies and the development of transgenic techniques provide the tools necessary to produce mosquito sexing strains, which promise to improve current malaria-control programs and pave the way for new ones. In this review, the progress made in the development of transgenic sexing strains for the control of Anopheles gambiae, a major vector of human malaria, is discussed.
Kyrou K, Hammond AM, Galizi R, et al., 2018, A CRISPR-Cas9 gene drive targeting doublesex causes complete population suppression in caged Anopheles gambiae mosquitoes, Nature Biotechnology, Vol: 36, Pages: 1062-1066, ISSN: 1087-0156
In the human malaria vector Anopheles gambiae, the gene doublesex (Agdsx) encodes two alternatively spliced transcripts, dsx-female (AgdsxF) and dsx-male (AgdsxM), that control differentiation of the two sexes. The female transcript, unlike the male, contains an exon (exon 5) whose sequence is highly conserved in all Anopheles mosquitoes so far analyzed. We found that CRISPR–Cas9-targeted disruption of the intron 4–exon 5 boundary aimed at blocking the formation of functional AgdsxF did not affect male development or fertility, whereas females homozygous for the disrupted allele showed an intersex phenotype and complete sterility. A CRISPR–Cas9 gene drive construct targeting this same sequence spread rapidly in caged mosquitoes, reaching 100% prevalence within 7–11 generations while progressively reducing egg production to the point of total population collapse. Owing to functional constraint of the target sequence, no selection of alleles resistant to the gene drive occurred in these laboratory experiments. Cas9-resistant variants arose in each generation at the target site but did not block the spread of the drive.
Hammond A, Karlsson X, Morianou I, et al., 2018, Regulation of gene drive expression increases invasive potential and mitigates resistance
<jats:title>Abstract</jats:title><jats:p>CRISPR-Cas9 nuclease-based gene drives rely on inducing chromosomal breaks in the germline that are repaired in ways that lead to a biased inheritance of the drive. Gene drives designed to impair female fertility can suppress populations of the mosquito vector of malaria. However, strong unintended fitness costs, due to ectopic nuclease expression, and high levels of resistant mutations, limited the potential of the first generation of gene drives to spread.</jats:p><jats:p>Here we show that changes to regulatory sequences in the drive element, designed to contain nuclease expression to the germline, confer improved fecundity over previous versions and generate drastically lower rates of target site resistance. We employed a genetic screen to show that this effect is explained by reduced rates of end-joining repair of DNA breaks at the target site caused by deposited nuclease in the embryo.</jats:p><jats:p>Highlighting the impact of deposited Cas9, many of the mutations arising from this source of nuclease activity in the embryo are heritable, thereby having the potential to generate resistant target sites that reduce the penetrance of the gene drive.</jats:p><jats:p>Finally, in cage invasion experiments these gene drives show improved invasion dynamics compared to first generation drives, resulting in greater than 90% suppression of the reproductive output and a delay in the emergence of target site resistance, even at a resistance-prone target sequence. We shed light on the dynamics of generation and selection of resistant alleles in a population by tracking, longitudinally, the frequency of resistant alleles in the face of an invading gene drive. Our results illustrate important considerations for future gene drive design and should expedite the development of gene drives robust to resistance.</jats:p>
Quinlan MM, Birungi J, Coulibaly MB, et al., 2018, Containment studies of transgenic mosquitoes in disease endemic countries: the broad concept of facilities readiness, Vector-Borne and Zoonotic Diseases, Vol: 18, Pages: 14-20, ISSN: 1530-3667
Genetic strategies for large scale pest or vector control using modified insects are not yet operational in Africa, and currently rely on import of the modified strains to begin preliminary, contained studies. Early involvement of research teams from participating countries is crucial to evaluate candidate field interventions. Following the recommended phased approach for novel strategies, evaluation should begin with studies in containment facilities. Experiences to prepare facilities and build international teams for research on transgenic mosquitoes revealed some important organizing themes underlying the concept of “facilities readiness,” or the point at which studies in containment may proceed, in sub-Saharan African settings. First, “compliance” for research with novel or non-native living organisms was defined as the fulfillment of all legislative and regulatory requirements. This is not limited to regulations regarding use of transgenic organisms. Second, the concept of “colony utility” was related to the characteristics of laboratory colonies being produced so that results of studies may be validated across time, sites, and strains or technologies; so that the appropriate candidate strains are moved forward toward field studies. Third, the importance of achieving “defensible science” was recognized, including that study conclusions can be traced back to evidence, covering the concerns of various stakeholders over the long term. This, combined with good stewardship of resources and appropriate funding, covers a diverse set of criteria for declaring when “facilities readiness” has been attained. It is proposed that, despite the additional demands on time and resources, only with the balance of and rigorous achievement of each of these organizing themes can collaborative research into novel strategies in vector or pest control reliably progress past initial containment studies.
Nolan T, 2017, How to handle gene drives in arthropods?, Pathog Glob Health, Vol: 111, Pages: 403-403
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