Imperial College London

DrTonyNolan

Faculty of Natural SciencesDepartment of Life Sciences

Honorary Senior Lecturer
 
 
 
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Contact

 

t.nolan

 
 
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Assistant

 

Mrs Lucy Collyns +44 (0)20 7594 5395

 
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Location

 

Sir Alexander Fleming BuildingSouth Kensington Campus

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Summary

 

Publications

Publication Type
Year
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69 results found

Quinn C, Anthousi A, Wondji C, Nolan Tet al., 2021, CRISPR-mediated knock-in of transgenes into the malaria vector Anopheles funestus, G3 Genes|Genomes|Genetics, Vol: 11

<jats:title>Abstract</jats:title> <jats:p>The ability to introduce mutations, or transgenes, of choice to precise genomic locations has revolutionized our ability to understand how genes and organisms work. In many mosquito species that are vectors of various human diseases, 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 Anopheles funestus 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. We describe herein an optimized 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 the 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 A. funestus.</jats:p>

Journal article

Hammond A, Pollegioni P, Persampieri T, North A, Minuz R, Trusso A, Bucci A, Kyrou K, Morianou I, Simoni A, Nolan T, Müller R, Crisanti Aet 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.

Journal article

Grigoraki L, Cowlishaw R, Nolan T, Donnelly M, Lycett G, Ranson Het 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

Journal article

O'Loughlin SM, Forster AJ, Fuchs S, Dottorini T, Nolan T, Crisanti A, Burt Aet 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

Journal article

Grigoraki L, Cowlishaw R, Nolan T, Donnelly M, Lycett G, Ranson Het al., 2021, CRISPR/Cas9 modified An. gambiae 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 &gt;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

Journal article

Hammond A, Pollegioni P, Persampieri T, North A, Minuz R, Trusso A, Bucci A, Kyrou K, Morianou I, Simoni A, Nolan T, Müller R, Crisanti Aet 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>

Journal article

Quinn C, Anthousi A, Wondji C, Nolan Tet al., 2021, CRISPR-mediated knock-in of transgenes into the malaria vector Anopheles funestus

<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>

Journal article

Ma Q, Srivastav SP, Gamez S, Dayama G, Feitosa-Suntheimer F, Patterson EI, Johnson RM, Matson EM, Gold AS, Brackney DE, Connor JH, Colpitts TM, Hughes GL, Rasgon JL, Nolan T, Akbari OS, Lau NCet al., 2021, A mosquito small RNA genomics resource reveals dynamic evolution and host responses to viruses and transposons., Genome Res, Vol: 31, Pages: 512-528

Although mosquitoes are major transmission vectors for pathogenic arboviruses, viral infection has little impact on mosquito health. This immunity is caused in part by mosquito RNA interference (RNAi) pathways that generate antiviral small interfering RNAs (siRNAs) and Piwi-interacting RNAs (piRNAs). RNAi also maintains genome integrity by potently repressing mosquito transposon activity in the germline and soma. However, viral and transposon small RNA regulatory pathways have not been systematically examined together in mosquitoes. Therefore, we developed an integrated mosquito small RNA genomics (MSRG) resource that analyzes the transposon and virus small RNA profiles in mosquito cell cultures and somatic and gonadal tissues across four medically important mosquito species. Our resource captures both somatic and gonadal small RNA expression profiles within mosquito cell cultures, and we report the evolutionary dynamics of a novel Mosquito-Conserved piRNA Cluster Locus (MCpiRCL) made up of satellite DNA repeats. In the larger culicine mosquito genomes we detected highly regular periodicity in piRNA biogenesis patterns coinciding with the expansion of Piwi pathway genes. Finally, our resource enables detection of cross talk between piRNA and siRNA populations in mosquito cells during a response to virus infection. The MSRG resource will aid efforts to dissect and combat the capacity of mosquitoes to tolerate and spread arboviruses.

Journal article

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'.

Journal article

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

Journal article

Kranjc N, Crisanti A, Nolan T, Bernardini Fet 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.

Journal article

Hammond A, Karlsson X, Morianou I, Kyrou K, Beaghton A, Gribble M, Kranjc N, Galizi R, Burt A, Crisanti A, Nolan Tet 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

Journal article

Simoni A, Hammond AM, Beaghton AK, Galizi R, Taxiarchi C, Kyrou K, Meacci D, Gribble M, Morselli G, Burt A, Nolan T, Crisanti Aet 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.

Journal article

Gomulski LM, Manni M, Carraretto D, Nolan T, Lawson D, Ribeiro JM, Malacrida AR, Gasperi Get 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

Journal article

Pollegioni P, North AR, Persampieri T, Bucci A, Minuz RL, Groneberg DA, Nolan T, Papathanos P-A, Crisanti A, Mueller Ret 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

Journal article

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

Journal article

James SL, Marshall JM, Christophides GK, Okumu FO, Nolan Tet 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.

Journal article

Beaghton AK, Hammond A, Nolan T, Crisanti A, Burt Aet 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.

Journal article

Taxiarchi C, Kranjc N, Kriezis A, Kyrou K, Bernardini F, Russell S, Nolan T, Crisanti A, Galizi Ret al., 2019, High-resolution transcriptional profiling of Anopheles gambiae spermatogenesis reveals mechanisms of sex chromosome regulation, SCIENTIFIC REPORTS, Vol: 9, ISSN: 2045-2322

Journal article

Bernardini F, Kriezis A, Galizi R, Nolan T, Crisanti Aet 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

Journal article

Bernardini F, Kriezis A, Galizi R, Nolan T, Crisanti Aet 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.

Journal article

Bernardini F, Haghighat-Khah RE, Galizi R, Hammond AM, Nolan T, Crisanti Aet 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.

Journal article

Kyrou K, Hammond AM, Galizi R, Kranjc N, Burt A, Beaghton AK, Nolan T, Crisanti Aet 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.

Journal article

Hammond A, Karlsson X, Morianou I, Kyrou K, Beaghton A, Gribble M, Kranjc N, Galizi R, Burt A, Crisanti A, Nolan Tet al., 2018, Regulation of gene drive expression increases invasive potential and mitigates resistance, Publisher: Cold Spring Harbor Laboratory

<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>

Working paper

Quinlan MM, Birungi J, Coulibaly MB, Diabate A, Facchinelli L, Mukabana WR, Mutunga JM, Nolan T, Raymond P, Traore SFet 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.

Journal article

Nolan T, 2017, How to handle gene drives in arthropods?, Pathog Glob Health, Vol: 111, Pages: 403-403

Journal article

Lombardo F, Salvemini M, Fiorillo C, Nolan T, Zwiebel LJ, Ribeiro JM, Arca Bet al., 2017, Deciphering the olfactory repertoire of the tiger mosquito Aedes albopictus, BMC Genomics, Vol: 18, ISSN: 1471-2164

BackgroundThe Asian tiger mosquito Aedes albopictus is a highly invasive species and competent vector of several arboviruses (e.g. dengue, chikungunya, Zika) and parasites (e.g. dirofilaria) of public health importance. Compared to other mosquito species, Ae. albopictus females exhibit a generalist host seeking as well as a very aggressive biting behaviour that are responsible for its high degree of nuisance. Several complex mosquito behaviours such as host seeking, feeding, mating or oviposition rely on olfactory stimuli that target a range of sensory neurons localized mainly on specialized head appendages such as antennae, maxillary palps and the mouthparts.ResultsWith the aim to describe the Ae. albopictus olfactory repertoire we have used RNA-seq to reveal the transcriptome profiles of female antennae and maxillary palps. Male heads and whole female bodies were employed as reference for differential expression analysis. The relative transcript abundance within each tissue (TPM, transcripts per kilobase per million) and the pairwise differential abundance in the different tissues (fold change values and false discovery rates) were evaluated. Contigs upregulated in the antennae (620) and maxillary palps (268) were identified and relative GO and PFAM enrichment profiles analysed. Chemosensory genes were described: overall, 77 odorant binding proteins (OBP), 82 odorant receptors (OR), 60 ionotropic receptors (IR) and 30 gustatory receptors (GR) were identified by comparative genomics and transcriptomics. In addition, orthologs of genes expressed in the female/male maxillary palps and/or antennae and involved in thermosensation (e.g. pyrexia and arrestin1), mechanosensation (e.g. piezo and painless) and neuromodulation were classified.ConclusionsWe provide here the first detailed transcriptome of the main Ae. albopictus sensory appendages, i.e. antennae and maxillary palps. A deeper knowledge of the olfactory repertoire of the tiger mosquito will help to better under

Journal article

Hammond AM, Kyrou K, Bruttini M, North A, Galizi R, Karlsson X, Kranjc N, Carpi FM, D'Aurizio R, Crisanti A, Nolan Tet al., 2017, The creation and selection of mutations resistant to a gene drive over multiple generations in the malaria mosquito, PLoS Genetics, Vol: 13, ISSN: 1553-7390

Gene drives have enormous potential for the control of insect populations of medical and agricultural relevance. By preferentially biasing their own inheritance, gene drives can rapidly introduce genetic traits even if these confer a negative fitness effect on the population. We have recently developed gene drives based on CRISPR nuclease constructs that are designed to disrupt key genes essential for female fertility in the malaria mosquito. The construct copies itself and the associated genetic disruption from one homologous chromosome to another during gamete formation, a process called homing that ensures the majority of offspring inherit the drive. Such drives have the potential to cause long-lasting, sustainable population suppression, though they are also expected to impose a large selection pressure for resistance in the mosquito. One of these population suppression gene drives showed rapid invasion of a caged population over 4 generations, establishing proof of principle for this technology. In order to assess the potential for the emergence of resistance to the gene drive in this population we allowed it to run for 25 generations and monitored the frequency of the gene drive over time. Following the initial increase of the gene drive we observed a gradual decrease in its frequency that was accompanied by the spread of small, nuclease-induced mutations at the target gene that are resistant to further cleavage and restore its functionality. Such mutations showed rates of increase consistent with positive selection in the face of the gene drive. Our findings represent the first documented example of selection for resistance to a synthetic gene drive and lead to important design recommendations and considerations in order to mitigate for resistance in future gene drive applications.

Journal article

Bernardini F, Galizi R, Wunderlich M, Taxiarchi C, Kranjc N, Kyrou K, Hammond A, Nolan T, Lawniczak MNK, Papathanos PA, Crisanti A, Windbichler Net al., 2017, Cross-Species Y Chromosome Function Between Malaria Vectors of the Anopheles gambiae Species Complex., Genetics, ISSN: 0016-6731

Y chromosome function, structure and evolution is poorly understood in many species including the Anopheles genus of mosquitoes, an emerging model system for studying speciation that also represents the major vectors of malaria. While the Anopheline Y had previously been implicated in male mating behavior, recent data from the Anopheles gambiae complex suggests that, apart from the putative primary sex-determiner, no other genes are conserved on the Y. Studying the functional basis of the evolutionary divergence of the Y chromosome in the gambiae complex is complicated by complete F1 male hybrid sterility. Here we used an F1xF0 crossing scheme to overcome a severe bottleneck of male hybrid incompatibilities and enabled us to experimentally purify a genetically labelled A. gambiae Y chromosome in an A. arabiensis background. Whole genome sequencing confirmed that the A. gambiae Y retained its original sequence content in the A. arabiensis genomic background. In contrast to comparable experiments in Drosophila, we find that the presence of a heterospecific Y chromosome has no significant effect on the expression of A. arabiensis genes and transcriptional differences can be explained almost exclusively as a direct consequence of transcripts arising from sequence elements present on the A. gambiae Y chromosome itself. We find that Y hybrids show no obvious fertility defects and no substantial reduction in male competitiveness. Our results demonstrate that, despite their radically different structure, Y chromosomes of these two species of the gambiae complex that diverged an estimated 1.85Myr ago function interchangeably, thus indicating that the Y chromosome does not harbor loci contributing to hybrid incompatibility. Therefore, Y chromosome gene flow between members of the gambiae complex is possible even at their current level of divergence. Importantly, this also suggests that malaria control interventions based on sex-distorting Y drive would be transferable, whethe

Journal article

Werther R, Hallinan JP, Lambert AR, Havens K, Pogson M, Jarjour J, Galizi R, Windbichler N, Crisanti A, Nolan T, Stoddard BLet al., 2017, Crystallographic analyses illustrate significant plasticity and efficient recoding of meganuclease target specificity, Nucleic Acids Research, Vol: 45, Pages: 8621-8634, ISSN: 0305-1048

The retargeting of protein–DNA specificity, outsideof extremely modular DNA binding proteins suchas TAL effectors, has generally proved to be quitechallenging. Here, we describe structural analysesof five different extensively retargeted variants of asingle homing endonuclease, that have been shownto function efficiently in ex vivo and in vivo applications.The redesigned proteins harbor mutationsat up to 53 residues (18%) of their amino acid sequence,primarily distributed across the DNA bindingsurface, making them among the most signifi-cantly reengineered ligand-binding proteins to date.Specificity is derived from the combined contributionsof DNA-contacting residues and of neighboringresidues that influence local structural organization.Changes in specificity are facilitated by theability of all those residues to readily exchange bothform and function. The fidelity of recognition is notprecisely correlated with the fraction or total numberof residues in the protein–DNA interface that areactually involved in DNA contacts, including directionalhydrogen bonds. The plasticity of the DNArecognitionsurface of this protein, which allows substantialretargeting of recognition specificity withoutrequiring significant alteration of the surroundingprotein architecture, reflects the ability of the correspondinggenetic elements to maintain mobility andpersistence in the face of genetic drift within potentialhost target sites.

Journal article

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