Imperial College London

DrNikolaiWindbichler

Faculty of Natural SciencesDepartment of Life Sciences

Lecturer
 
 
 
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Contact

 

+44 (0)20 7594 1650n.windbichler

 
 
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Location

 

Sir Alexander Fleming BuildingSouth Kensington Campus

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Summary

 

Publications

Publication Type
Year
to

45 results found

Hoermann A, Tapanelli S, Capriotti P, Masters EKG, Habtewold T, Christophides GK, Windbichler Net al., 2020, Converting endogenous genes of the malaria mosquito into simple non-autonomous gene drives for population replacement

<jats:title>Abstract</jats:title><jats:p>Gene drives for mosquito population replacement are promising tools for malaria control. However, there is currently no clear pathway for safely testing such tools in endemic countries. The lack of well-characterized promoters for infection-relevant tissues and regulatory hurdles are further obstacles for their design and use. Here we explore how minimal genetic modifications of endogenous mosquito genes can convert them directly into non-autonomous gene drives without disrupting their expression. We co-opted the native regulatory sequences of three midgut-specific loci of the malaria vector <jats:italic>Anopheles gambiae</jats:italic> to host a prototypical antimalarial molecule and guide-RNAs encoded within artificial introns, that support efficient gene drive. We assess the propensity of these modifications to interfere with the development of <jats:italic>Plasmodium falciparum</jats:italic> and their effect on fitness. Because of their inherent simplicity and passive mode of drive such traits could form part of an accepted testing pathway of gene drives for malaria eradication.</jats:p>

Journal article

Miguel-Aliaga I, Redhai S, Pilgrim C, Gaspar P, van Giesen L, Lopes T, Riabinina O, Grenier T, Milona A, Chanana B, Swadling J, Wang Y-F, Dahalan F, Yuan M, Wilsch-Brauninger M, Lin W-H, Dennison N, Capriotti P, Lawniczak M, Baines R, Warnecke T, Windbichler N, Leulier F, Bellono Net al., 2020, An intestinal zinc sensor regulates food intake and developmental growth, Nature, Vol: 580, Pages: 263-268, ISSN: 0028-0836

In cells, organs and whole organisms, nutrient sensing is key to maintaining homeostasis and adapting to a fluctuating environment1. In many animals, nutrient sensors are found within the enteroendocrine cells of the digestive system; however, less is known about nutrient sensing in their cellular siblings, the absorptive enterocytes1. Here we use a genetic screen in Drosophila melanogaster to identify Hodor, an ionotropic receptor in enterocytes that sustains larval development, particularly in nutrient-scarce conditions. Experiments in Xenopus oocytes and flies indicate that Hodor is a pH-sensitive, zinc-gated chloride channel that mediates a previously unrecognized dietary preference for zinc. Hodor controls systemic growth from a subset of enterocytes—interstitial cells—by promoting food intake and insulin/IGF signalling. Although Hodor sustains gut luminal acidity and restrains microbial loads, its effect on systemic growth results from the modulation of Tor signalling and lysosomal homeostasis within interstitial cells. Hodor-like genes are insect-specific, and may represent targets for the control of disease vectors. Indeed, CRISPR–Cas9 genome editing revealed that the single hodor orthologue in Anopheles gambiae is an essential gene. Our findings highlight the need to consider the instructive contributions of metals—and, more generally, micronutrients—to energy homeostasis.

Journal article

Fasulo B, Meccariello A, Morgan M, Borufka C, Papathanos PA, Windbichler Net al., 2020, A fly model establishes distinct mechanisms for synthetic CRISPR/Cas9 sex distorters, PLOS GENETICS, Vol: 16, ISSN: 1553-7404

Journal article

Dahalan FA, Churcher TS, Windbichler N, Lawniczak MKNet al., 2019, The male mosquito contribution towards malaria transmission: Mating influences the Anopheles female midgut transcriptome and increases female susceptibility to human malaria parasites., PLoS Pathogens, Vol: 15, Pages: 1-19, ISSN: 1553-7366

Mating causes dramatic changes in female physiology, behaviour, and immunity in many insects, inducing oogenesis, oviposition, and refractoriness to further mating. Females from the Anopheles gambiae species complex typically mate only once in their lifetime during which they receive sperm and seminal fluid proteins as well as a mating plug that contains the steroid hormone 20-hydroxyecdysone. This hormone, which is also induced by blood-feeding, plays a major role in activating vitellogenesis for egg production. Here we show that female Anopheles coluzzii susceptibility to Plasmodium falciparum infection is significantly higher in mated females compared to virgins. We also find that mating status has a major impact on the midgut transcriptome, detectable only under sugar-fed conditions: once females have blood-fed, the transcriptional changes that are induced by mating are likely masked by the widespread effects of blood-feeding on gene expression. To determine whether increased susceptibility to parasites could be driven by the additional 20E that mated females receive from males, we mimicked mating by injecting virgin females with 20E, finding that these females are significantly more susceptible to human malaria parasites than virgin females injected with the control 20E carrier. Further RNAseq was carried out to examine whether the genes that change upon 20E injection in the midgut are similar to those that change upon mating. We find that 79 midgut-expressed genes are regulated in common by both mating and 20E, and 96% (n = 76) of these are regulated in the same direction (up vs down in 20E/mated). Together, these findings show that male Anopheles mosquitoes induce changes in the female midgut that can affect female susceptibility to P. falciparum. This implies that in nature, males might contribute to malaria transmission in previously unappreciated ways, and that vector control strategies that target males may have additional benefits towards reducing transm

Journal article

Giese B, Friess JL, Barton NH, Messer PW, Debarre F, Schetelig MF, Windbichler N, Meimberg H, Boete Cet al., 2019, Gene Drives: Dynamics and Regulatory Matters-A Report from the Workshop "Evaluation of Spatial and Temporal Control of Gene Drives," April 4-5, 2019, Vienna, BIOESSAYS, Vol: 41, ISSN: 0265-9247

Journal article

Meccariello A, Salvemini M, Primo P, Hall B, Koskiniot P, Dalikova M, Gravina A, Gucciar MA, Forlenza F, Gregoriou ME, Ippolito D, Monti SM, Petrella V, Perrotta MM, Schmeing S, Ruggiero A, Scolari F, Giordano E, Tsoumani KT, Marec F, Windbichler N, Nagaraju J, Arunkumar KP, Bourtzis K, Mathiopoulos KD, Ragoussis J, Vitagliano L, Tu Z, Papathanos PA, Robinson MD, Saccone Get al., 2019, Maleness-on-the-Y (MoY) orchestrates male sex determination in major agricultural fruit fly pests, SCIENCE, Vol: 365, Pages: 1457-+, ISSN: 0036-8075

Journal article

Habtewold T, Tapanelli S, Ellen KG M, Astrid H, Nikolai W, George K Cet al., 2019, Streamlined SMFA and mosquito dark-feeding regime significantly improve malaria transmission-blocking assay robustness and sensitivity, Malaria Journal, Vol: 18, ISSN: 1475-2875

BackgroundThe development of malaria transmission-blocking strategies including the generation of malaria refractory mosquitoes to replace the wild populations through means of gene drives hold great promise. The standard membrane feeding assay (SMFA) that involves mosquito feeding on parasitized blood through an artificial membrane system is a vital tool for evaluating the efficacy of transmission-blocking interventions. However, despite the availability of several published protocols, the SMFA remains highly variable and broadly insensitive.MethodsThe SMFA protocol was optimized through coordinated culturing of Anopheles coluzzii mosquitoes and Plasmodium falciparum parasite coupled with placing mosquitoes under a strict dark regime before, during, and after the gametocyte feed.ResultsA detailed description of essential steps is provided toward synchronized generation of highly fit An. coluzzii mosquitoes and P. falciparum gametocytes in preparation for an SMFA. A dark-infection regime that emulates the natural vector-parasite interaction system is described, which results in a significant increase in the infection intensity and prevalence. Using this optimal SMFA pipeline, a series of putative transmission-blocking antimicrobial peptides (AMPs) were screened, confirming that melittin and magainin can interfere with P. falciparum development in the vector.ConclusionA robust SMFA protocol that enhances the evaluation of interventions targeting human malaria transmission in laboratory setting is reported. Melittin and magainin are identified as highly potent antiparasitic AMPs that can be used for the generation of refractory Anopheles gambiae mosquitoes.

Journal article

Nash A, Urdaneta Mignini G, Beaghton A, Hoermann A, Papathanos P, Christophides G, Windbichler Net al., 2019, Integral Gene Drives for population replacement, Biology Open, Vol: 8, ISSN: 2046-6390

A first generation of CRISPR-based gene drives has now been tested in the laboratory in a number of organisms, including malaria vector mosquitoes. Challenges for their use in the area-wide genetic control of vector-borne disease have been identified, including the development of target site resistance, their long-term efficacy in the field, their molecular complexity, and practical and legal limitations for field testing of both gene drive and coupled anti-pathogen traits. We have evaluated theoretically the concept of integral gene drive (IGD) as an alternative paradigm for population replacement. IGDs incorporate a minimal set of molecular components, including drive and anti-pathogen effector elements directly embedded within endogenous genes – an arrangement that in theory allows targeting functionally conserved coding sequences without disrupting their function. Autonomous and non-autonomous IGD strains could be generated, optimized, regulated and imported independently. We performed quantitative modeling comparing IGDs with classical replacement drives and show that selection for the function of the hijacked host gene can significantly reduce the establishment of resistant alleles in the population, while drive occurring at multiple genomic loci prolongs the duration of transmission blockage in the face of pre-existing target site variation. IGD thus has potential as a more durable and flexible population replacement strategy.

Journal article

Waters AJ, Capriotti P, Gaboriau DCA, Papathanos PA, Windbichler Net al., 2018, Rationally-engineered reproductive barriers using CRISPR & CRISPRa: an evaluation of the synthetic species concept in Drosophila melanogaster, Scientific Reports, Vol: 8, ISSN: 2045-2322

The ability to erect rationally-engineered reproductive barriers in animal or plant species promises to enable a number of biotechnological applications such as the creation of genetic firewalls, the containment of gene drives or novel population replacement and suppression strategies for genetic control. However, to date no experimental data exist that explores this concept in a multicellular organism. Here we examine the requirements for building artificial reproductive barriers in the metazoan model Drosophila melanogaster by combining CRISPR-based genome editing and transcriptional transactivation (CRISPRa) of the same loci. We directed 13 single guide RNAs (sgRNAs) to the promoters of 7 evolutionary conserved genes and used 11 drivers to conduct a misactivation screen. We identify dominant-lethal activators of the eve locus and find that they disrupt development by strongly activating eve outside its native spatio-temporal context. We employ the same set of sgRNAs to isolate, by genome editing, protective INDELs that render these loci resistant to transactivation without interfering with target gene function. When these sets of genetic components are combined we find that complete synthetic lethality, a prerequisite for most applications, is achievable using this approach. However, our results suggest a steep trade-off between the level and scope of dCas9 expression, the degree of genetic isolation achievable and the resulting impact on fly fitness. The genetic engineering strategy we present here allows the creation of single or multiple reproductive barriers and could be applied to other multicellular organisms such as disease vectors or transgenic organisms of economic importance.

Journal article

Nash A, Urdaneta GM, K Beaghton A, Hoermann A, Aris Papathanos P, Christophides GK, Windbichler Net al., 2018, Integral Gene Drives: an “operating system” for population replacement

<jats:title>Abstract</jats:title><jats:p>First generation CRISPR-based gene drives have now been tested in the laboratory in a number of organisms including malaria vector mosquitoes. A number of challenges for their use in the area-wide genetic control of vector-borne disease have been identified. These include the development of target site resistance, their long-term efficacy in the field, their molecular complexity, and the practical and legal limitations for field testing of both gene drive and coupled anti-pathogen traits. To address these challenges, we have evaluated the concept of Integral Gene Drive (IGD) as an alternative paradigm for population replacement. IGDs incorporate a minimal set of molecular components, including both the drive and the anti-pathogen effector elements directly embedded within endogenous genes – an arrangement which we refer to as gene “hijacking”. This design would allow autonomous and non-autonomous IGD traits and strains to be generated, tested, optimized, regulated and imported independently. We performed quantitative modelling comparing IGDs with classical replacement drives and show that selection for the function of the hijacked host gene can significantly reduce the establishment of resistant alleles in the population while hedging drive over multiple genomic loci prolongs the duration of transmission blockage in the face of pre-existing target-site variation. IGD thus has the potential to yield more durable and flexible population replacement traits.</jats:p>

Journal article

Waters AJ, Capriotti P, Gaboriau D, Papathanos PA, Windbichler Net al., 2018, Rationally-engineered reproductive barriers using CRISPR & CRISPRa: an evaluation of the synthetic species concept inDrosophila melanogaster, Publisher: bioRxiv

The ability to erect rationally-engineered reproductive barriers in animal or plant species promises to enable a number of biotechnological applications such as the creation of genetic firewalls, the containment of gene drives or novel population replacement and suppression strategies for genetic control. However, to date no experimental data exist that explores this concept in a multicellular organism. Here we examine the requirements for building artificial reproductive barriers in the metazoan model Drosophila melanogaster by combining CRISPR-based genome editing and transcriptional transactivation (CRISPRa) of the same loci. We directed 13 single guide RNAs (sgRNAs) to the promoters of 7 evolutionary conserved genes and used 11 drivers to conduct a miss-activation screen. We identify dominant-lethal activators of the eve locus and find that they disrupt development by strongly activating eve outside its native spatio-temporal context. We employ the same set of sgRNAs to isolate, by genome editing, protective INDELs that render these loci resistant to transactivation without interfering with target gene function. When these sets of genetic components are combined we find that complete synthetic lethality, a prerequisite for most applications, is achievable using this approach. However, our results suggest a steep trade-off between the level and scope of dCas9 expression, the degree of genetic isolation achievable and the resulting impact on fly fitness. The genetic engineering strategy we present here allows the creation of single or multiple reproductive barriers and could be applied to other multicellular organisms such as disease vectors or transgenic organisms of economic importance.

Working paper

Papathanos PA, Windbichler N, 2018, Redkmer: An assembly-free pipeline for the identification of abundant and specific X-chromosome target sequences for X-shredding by CRISPR endonucleases, The Crispr Journal, Vol: 1, Pages: 88-98, ISSN: 2573-1599

CRISPR-based synthetic sex ratio distorters, which operate by shredding the X-chromosome during male meiosis, are promising tools for the area-wide control of harmful insect pest or disease vector species. X-shredders have been proposed as tools to suppress insect populations by biasing the sex ratio of the wild population toward males, thus reducing its natural reproductive potential. However, to build synthetic X-shredders based on CRISPR, the selection of gRNA targets, in the form of high-copy sequence repeats on the X chromosome of a given species, is difficult, since such repeats are not accurately resolved in genome assemblies and cannot be assigned to chromosomes with confidence. We have therefore developed the redkmer computational pipeline, designed to identify short and highly abundant sequence elements occurring uniquely on the X chromosome. Redkmer was designed to use as input minimally processed whole genome sequence data from males and females. We tested redkmer with short- and long-read whole genome sequence data of Anopheles gambiae, the major vector of human malaria, in which the X-shredding paradigm was originally developed. Redkmer established long reads as chromosomal proxies with excellent correlation to the genome assembly and used them to rank X-candidate kmers for their level of X-specificity and abundance. Among these, a high-confidence set of 25-mers was identified, many belonging to previously known X-chromosome repeats of Anopheles gambiae, including the ribosomal gene array and the selfish elements harbored within it. Data from a control strain, in which these repeats are shared with the Y chromosome, confirmed the elimination of these kmers during filtering. Finally, we show that redkmer output can be linked directly to gRNA selection and off-target prediction. In addition, the output of redkmer, including the prediction of chromosomal origin of single-molecule long reads and chromosome specific kmers, could also be used for the charact

Journal article

Papathanos PA, Windbichler N, 2017, redkmer: an assembly-free pipeline for the identification of abundant and specific X-chromosome target sequences for X-shredding by CRISPR endonucleases

<jats:title>Abstract</jats:title><jats:p>CRISPR-based synthetic sex ratio distorters, that operate by shredding the X-chromosome during male meiosis, are promising tools for the area-wide control of harmful insect pest or disease vector species. However, the selection of gRNA targets, in the form of high-copy sequence repeats on the X chromosome of a given species, is difficult since such repeats are not accurately resolved in genome assemblies and can’t be assigned to chromosomes with confidence. We have therefore developed the redkmer computational pipeline, designed to identify short and highly-abundant sequence elements occurring uniquely on the X-chromosome. Redkmer was designed to use as input exclusively raw WGS data from males and females. We tested redkmer with suitable short and long read WGS data of <jats:italic>An. gambiae</jats:italic>, the major vector of human malaria, in which the X-shredding paradigm was originally developed. Redkmer establishes long reads as chromosomal proxies with excellent correlation to the genome assembly and uses them to rank X-candidate kmers for their level of X-specificity and abundance. Redkmer identified a high-confidence set of 25-mers, many of which belong to previously known X-chromosome specific repeats of <jats:italic>An. gambiae</jats:italic>, including the ribosomal gene array and the selfish genetics elements harbored within it. WGS data from a control strain in which these repeats are also present on the Y chromosome confirmed the elimination of these kmers in the filtering steps. Finally, we show that redkmer output can be linked directly to gRNA selection and can also inform gRNA off-target prediction. The redkmer pipeline is designed to enable the generation of synthetic sex ratio distorters for the control of harmful insect species of medical or agricultural importance. It proceeds from WGS input data to deliver candidate X-specific CRISPR gRNA candidate target

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

Papa F, Windbichler N, Waterhouse RM, Cagnetti A, D'Amato R, Persampieri T, Lawniczak MKN, Nolan T, Papathanos PAet al., 2017, Rapid evolution of female-biased genes among four species of Anopheles malaria mosquitoes, GENOME RESEARCH, Vol: 27, Pages: 1536-1548, ISSN: 1088-9051

Understanding how phenotypic differences between males and females arise from the sex-biased expression of nearly identical genomes can reveal important insights into the biology and evolution of a species. Among Anopheles mosquito species, these phenotypic differences include vectorial capacity, as it is only females that blood feed and thus transmit human malaria. Here, we use RNA-seq data from multiple tissues of four vector species spanning the Anopheles phylogeny to explore the genomic and evolutionary properties of sex-biased genes. We find that, in these mosquitoes, in contrast to what has been found in many other organisms, female-biased genes are more rapidly evolving in sequence, expression, and genic turnover than male-biased genes. Our results suggest that this atypical pattern may be due to the combination of sex-specific life history challenges encountered by females, such as blood feeding. Furthermore, female propensity to mate only once in nature in male swarms likely diminishes sexual selection of post-reproductive traits related to sperm competition among males. We also develop a comparative framework to systematically explore tissue- and sex-specific splicing to document its conservation throughout the genus and identify a set of candidate genes for future functional analyses of sex-specific isoform usage. Finally, our data reveal that the deficit of male-biased genes on the X Chromosomes in Anopheles is a conserved feature in this genus and can be directly attributed to chromosome-wide transcriptional regulation that de-masculinizes the X in male reproductive tissues.

Journal article

Galizi R, Hammond A, Kyrou K, Taxiarchi C, Bernardini F, O'Loughlin SM, Papathanos PA, Nolan T, Windbichler N, Crisanti Aet al., 2016, A CRISPR-Cas9 sex-ratio distortion system for genetic control., Scientific Reports, Vol: 6, ISSN: 2045-2322

Genetic control aims to reduce the ability of insect pest populations to cause harm via the release of modified insects. One strategy is to bias the reproductive sex ratio towards males so that a population decreases in size or is eliminated altogether due to a lack of females. We have shown previously that sex ratio distortion can be generated synthetically in the main human malaria vector Anopheles gambiae, by selectively destroying the X-chromosome during spermatogenesis, through the activity of a naturally-occurring endonuclease that targets a repetitive rDNA sequence highly-conserved in a wide range of organisms. Here we describe a CRISPR-Cas9 sex distortion system that targets ribosomal sequences restricted to the member species of the Anopheles gambiae complex. Expression of Cas9 during spermatogenesis resulted in RNA-guided shredding of the X-chromosome during male meiosis and produced extreme male bias among progeny in the absence of any significant reduction in fertility. The flexibility of CRISPR-Cas9 combined with the availability of genomic data for a range of insects renders this strategy broadly applicable for the species-specific control of any pest or vector species with an XY sex-determination system by targeting sequences exclusive to the female sex chromosome.

Journal article

Hall AB, Papathanos P-A, Sharma A, Cheng C, Akbari OS, Assour L, Bergman NH, Cagnetti A, Crisanti A, Dottorini T, Fiorentini E, Galizi R, Hnath J, Jiang X, Koren S, Nolan T, Radune D, Sharakhova MV, Steele A, Timoshevskiy VA, Windbichler N, Zhang S, Hahn MW, Phillippy AM, Emrich SJ, Sharakhov IV, Tu ZJ, Besansky NJet al., 2016, Radical remodeling of the Y chromosome in a recent radiation of malaria mosquitoes, Proceedings of the National Academy of Sciences of the United States of America, Vol: 113, Pages: E2114-E2123

Journal article

Hammond A, Galizi R, Kyrou K, Simoni A, Siniscalchi C, Katsanos D, Gribble M, Baker D, Marois E, Russell S, Burt A, Windbichler N, Crisanti A, Nolan Tet al., 2015, A CRISPR-Cas9 gene drive system-targeting female reproduction in the malaria mosquito vector Anopheles gambiae, Nature Biotechnology, Vol: 34, Pages: 78-83, ISSN: 1087-0156

Gene drive systems that enable super-Mendelian inheritance of a transgene have the potential to modify insect populations over a timeframe of a few years. We describe CRISPR-Cas9 endonuclease constructs that function as gene drive systems in Anopheles gambiae, the main vector for malaria. We identified three genes (AGAP005958, AGAP011377 and AGAP007280) that confer a recessive female-sterility phenotype upon disruption, and inserted into each locus CRISPR-Cas9 gene drive constructs designed to target and edit each gene. For each targeted locus we observed a strong gene drive at the molecular level, with transmission rates to progeny of 91.4 to 99.6%. Population modeling and cage experiments indicate that a CRISPR-Cas9 construct targeting one of these loci, AGAP007280, meets the minimum requirement for a gene drive targeting female reproduction in an insect population. These findings could expedite the development of gene drives to suppress mosquito populations to levels that do not support malaria transmission.

Journal article

Dritsou V, Topalis P, Windbichler N, Simoni A, Hall A, Lawson D, Hinsley M, Hughes D, Napolioni V, Crucianelli F, Deligianni E, Gasperi G, Gomulski LM, Savini G, Manni M, Scolari F, Malacrida AR, Arca B, Ribeiro JM, Lombardo F, Saccone G, Salvemini M, Moretti R, Aprea G, Calvitti M, Picciolini M, Papathanos PA, Spaccapelo R, Favia G, Crisanti A, Louis Cet al., 2015, A draft genome sequence of an invasive mosquito: an Italian Aedes albopictus, PATHOGENS AND GLOBAL HEALTH, Vol: 109, Pages: 207-220, ISSN: 2047-7724

Journal article

Simoni A, Siniscalchi C, Chan YS, Huen DS, Russell S, Windbichler N, Crisanti Aet al., 2015, Development of synthetic selfish elements based on modular nucleases in Drosophila melanogaster (vol 42, pg 7461, 2014), NUCLEIC ACIDS RESEARCH, Vol: 43, Pages: 2991-2991, ISSN: 0305-1048

Journal article

Volohonsky G, Terenzi O, Soichot J, Naujoks DA, Nolan T, Windbichler N, Kapps D, Smidler AL, Vittu A, Costa G, Steinert S, Levashina EA, Blandin SA, Marois Eet al., 2015, Tools for Anopheles gambiae Transgenesis, G3-Genes Genomes Genetics, Vol: 5, Pages: 1151-1163

Journal article

Simoni A, Siniscalchi C, Chan Y-S, Huen DS, Russell S, Windbichler N, Crisanti Aet al., 2014, Development of synthetic selfish elements based on modular nucleases in Drosophila melanogaster, Nucleic Acids Research, Vol: 42, Pages: 7461-7472, ISSN: 1362-4962

Selfish genes are DNA elements that increase their rate of genetic transmission at the expense of other genes in the genome and can therefore quickly spread within a population. It has been suggested that selfish elements could be exploited to modify the genome of entire populations for medical and ecological applications. Here we report that transcription activator-like effector nuclease (TALEN) and zinc finger nuclease (ZFN) can be engineered into site-specific synthetic selfish elements (SSEs) and demonstrate their transmission of up to 70% in the Drosophila germline. We show here that SSEs can spread via DNA break-induced homologous recombination, a process known as ‘homing’ similar to that observed for homing endonuclease genes (HEGs), despite their fundamentally different modes of DNA binding and cleavage. We observed that TALEN and ZFN have a reduced capability of secondary homing compared to HEG as their repetitive structure had a negative effect on their genetic stability. The modular architecture of ZFNs and TALENs allows for the rapid design of novel SSEs against specific genomic sequences making them potentially suitable for the genetic engineering of wild-type populations of animals and plants, in applications such as gene replacement or population suppression of pest species.

Journal article

Galizi R, Doyle LA, Menichelli M, Bernardini F, Deredec A, Burt A, Stoddard BL, Windbichler N, Crisanti Aet al., 2014, A synthetic sex ratio distortion system for the control of the human malaria mosquito, Nature Communications, Vol: 5, ISSN: 2041-1723

It has been theorized that inducing extreme reproductive sex ratios could be a method to suppress or eliminate pest populations. Limited knowledge about the genetic makeup and mode of action of naturally occurring sex distorters and the prevalence of co-evolving suppressors has hampered their use for control. Here we generate a synthetic sex distortion system by exploiting the specificity of the homing endonuclease I-PpoI, which is able to selectively cleave ribosomal gene sequences of the malaria vector Anopheles gambiae that are located exclusively on the mosquito’s X chromosome. We combine structure-based protein engineering and molecular genetics to restrict the activity of the potentially toxic endonuclease to spermatogenesis. Shredding of the paternal X chromosome prevents it from being transmitted to the next generation, resulting in fully fertile mosquito strains that produce >95% male offspring. We demonstrate that distorter male mosquitoes can efficiently suppress caged wild-type mosquito populations, providing the foundation for a new class of genetic vector control strategies.

Journal article

Bernardini F, Galizi R, Menichelli M, Papathanos P-A, Dritsou V, Marois E, Crisanti A, Windbichler Net al., 2014, Site-specific genetic engineering of the Anopheles gambiae Y chromosome, PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, Vol: 111, Pages: 7600-7605, ISSN: 0027-8424

Journal article

Simoni A, Siniscalchi C, Chan YS, Huen DS, Russell S, Windbichler N, Crisanti Aet al., 2013, DEVELOPMENT OF NOVEL SYNTHETIC SELFISH ELEMENTS FOR POPULATION ENGINEERING AND VECTOR CONTROL, PATHOGENS AND GLOBAL HEALTH, Vol: 107, Pages: 442-442, ISSN: 2047-7724

Journal article

Windbichler N, Galizi R, Crisanti A, 2013, A SYNTHETIC SEX DISTORTION SYSTEM IN ANOPHELES GAMBIAE, PATHOGENS AND GLOBAL HEALTH, Vol: 107, Pages: 440-441, ISSN: 2047-7724

Journal article

Galizi R, Windbichler N, Crisanti A, 2013, A SYNTHETIC SEX DISTORTER AS A NOVEL MALARIA VECTOR CONTROL STRATEGY, PATHOGENS AND GLOBAL HEALTH, Vol: 107, Pages: 445-445, ISSN: 2047-7724

Journal article

Windbichler N, Galizzi R, Burt A, Crisanti Aet al., 2012, Engineering mosquito population for vector control, Malaria Journal, Vol: 11

Journal article

Magnusson K, Lycett GJ, Mendes AM, Lynd A, Papathanos P-A, Crisanti A, Windbichler Net al., 2012, Demasculinization of the Anopheles gambiae X chromosome, BMC Evolutionary Biology, Vol: 12, ISSN: 1471-2148

BackgroundIn a number of organisms sex-biased genes are non-randomly distributed between autosomes and the shared sex chromosome X (or Z). Studies on Anopheles gambiae have produced conflicting results regarding the underrepresentation of male-biased genes on the X chromosome and it is unclear to what extent sexual antagonism, dosage compensation or X-inactivation in the male germline, the evolutionary forces that have been suggested to affect the chromosomal distribution of sex-biased genes, are operational in Anopheles.ResultsWe performed a meta-analysis of sex-biased gene expression in Anopheles gambiae which provides evidence for a general underrepresentation of male-biased genes on the X-chromosome that increased in significance with the observed degree of sex-bias. A phylogenomic comparison between Drosophila melanogaster, Aedes aegypti and Culex quinquefasciatus also indicates that the Anopheles X chromosome strongly disfavours the evolutionary conservation of male-biased expression and that novel male-biased genes are more likely to arise on autosomes. Finally, we demonstrate experimentally that transgenes situated on the Anopheles gambiae X chromosome are transcriptionally silenced in the male germline.ConclusionThe data presented here support the hypothesis that the observed demasculinization of the Anopheles X chromosome is driven by X-chromosome inactivation in the male germline and by sexual antagonism. The demasculinization appears to be the consequence of a loss of male-biased expression, rather than a failure in the establishment or the extinction of male-biased genes.

Journal article

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