Publications
62 results found
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 s
Bernardini F, Galizi R, Wunderlich M, et 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
Werther R, Hallinan JP, Lambert AR, et 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.
Papa F, Windbichler N, Waterhouse RM, et 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.
Galizi R, Hammond A, Kyrou K, et 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.
Hall AB, Papathanos P-A, Sharma A, et 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
Hammond A, Galizi R, Kyrou K, et al., 2016, 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.
Dritsou V, Topalis P, Windbichler N, et al., 2015, A draft genome sequence of an invasive mosquito: an Italian <i>Aedes</i> <i>albopictus</i>, PATHOGENS AND GLOBAL HEALTH, Vol: 109, Pages: 207-220, ISSN: 2047-7724
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- Citations: 25
Simoni A, Siniscalchi C, Chan YS, et 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
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- Citations: 1
Volohonsky G, Terenzi O, Soichot J, et al., 2015, Tools for Anopheles gambiae Transgenesis, G3-Genes Genomes Genetics, Vol: 5, Pages: 1151-1163
Simoni A, Siniscalchi C, Chan Y-S, et 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.
Galizi R, Doyle LA, Menichelli M, et 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.
Bernardini F, Galizi R, Menichelli M, et 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
Despite its function in sex determination and its role in driving genome evolution, the Y chromosome remains poorly understood in most species. Y chromosomes are gene-poor, repeat-rich and largely heterochromatic and therefore represent a difficult target for genetic engineering. The Y chromosome of the human malaria vector Anopheles gambiae appears to be involved in sex determination although very little is known about both its structure and function. Here, we characterize a transgenic strain of this mosquito species, obtained by transposon-mediated integration of a transgene construct onto the Y chromosome. Using meganuclease-induced homologous repair we introduce a site-specific recombination signal onto the Y chromosome and show that the resulting docking line can be used for secondary integration. To demonstrate its utility, we study the activity of a germ-line–specific promoter when located on the Y chromosome. We also show that Y-linked fluorescent transgenes allow automated sex separation of this important vector species, providing the means to generate large single-sex populations. Our findings will aid studies of sex chromosome function and enable the development of male-exclusive genetic traits for vector control.
Papathanos PA, Windbichler N, Akbari OS, 2014, Sex Ratio Manipulation for Insect Population Control, TRANSGENIC INSECTS: TECHNIQUES AND APPLICATIONS, Editors: Benedict, Publisher: CABI PUBLISHING-C A B INT, Pages: 83-100
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- Citations: 8
Simoni A, Siniscalchi C, Chan YS, et 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
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
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
Windbichler N, Galizzi R, Burt A, et al., 2012, Engineering mosquito population for vector control, Malaria Journal, Vol: 11
Magnusson K, Lycett GJ, Mendes AM, et 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.
Klein TA, Windbichler N, Deredec A, et al., 2012, Infertility resulting from transgenic I-PpoI male <i>Anopheles gambiae</i> in large cage trials, PATHOGENS AND GLOBAL HEALTH, Vol: 106, Pages: 20-31, ISSN: 2047-7724
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- Citations: 38
Windbichler N, 2012, Big insights from a small outbreak, PATHOGENS AND GLOBAL HEALTH, Vol: 106, Pages: 63-63, ISSN: 2047-7724
Windbichler N, Menichelli M, Papathanos PA, et al., 2011, A synthetic homing endonuclease-based gene drive system in the human malaria mosquito, NATURE, Vol: 473, Pages: 212-+, ISSN: 0028-0836
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- Citations: 244
Nolan T, Papathanos P, Windbichler N, et al., 2011, Developing transgenic Anopheles mosquitoes for the sterile insect technique, Genetica, Vol: 139, Pages: 33-39
Magnusson K, Mendes AM, Windbichler N, et al., 2011, Transcription Regulation of Sex-Biased Genes during Ontogeny in the Malaria Vector Anopheles gambiae, Plos One, Vol: 6
Papathanos PA, Windbichler N, Menichelli M, et al., 2009, The <i>vasa</i> regulatory region mediates germline expression and maternal transmission of proteins in the malaria mosquito <i>Anopheles gambiae</i>: a versatile tool for genetic control strategies, BMC MOLECULAR BIOLOGY, Vol: 10, ISSN: 1471-2199
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- Citations: 67
Windbichler N, Papathanos PA, Crisanti A, 2008, Targeting the X Chromosome during Spermatogenesis Induces Y Chromosome Transmission Ratio Distortion and Early Dominant Embryo Lethality in <i>Anopheles gambiae</i>, PLOS GENETICS, Vol: 4, ISSN: 1553-7404
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- Citations: 124
Windbichler N, von Pelchrzim F, Mayer O, et al., 2008, Isolation of small RNA-binding proteins from <i>E</i>. <i>coli</i> -: Evidence for frequent interaction of RNAs with RNA polymerase, RNA BIOLOGY, Vol: 5, Pages: 30-40, ISSN: 1547-6286
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- Citations: 43
Windbichler N, Papathanos PA, Catteruccia F, et al., 2007, Homing endonuclease mediated gene targeting in <i>Anopheles gambiae</i> cells and embryos, NUCLEIC ACIDS RESEARCH, Vol: 35, Pages: 5922-5933, ISSN: 0305-1048
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- Citations: 93
Dangerfield JA, Windbichler N, Salmons B, et al., 2006, Enhancement of the StreptoTag method for isolation of endogenously expressed proteins with complex RNA binding targets, ELECTROPHORESIS, Vol: 27, Pages: 1874-1877, ISSN: 0173-0835
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- Citations: 19
Windbichler N, Schroeder R, 2006, Isolation of specific RNA-binding proteins using the streptomycin-binding RNA aptamer, NATURE PROTOCOLS, Vol: 1, Pages: 638-U4, ISSN: 1754-2189
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- Citations: 41
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