Biology subjects

Windbichler, N.

Publications and source records attributed to Windbichler, N..

5 recordsLinked to original sources

bioRxiv2018

Integral Gene Drives: an \"operating system\" for population replacement

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.

synthetic biology↗

bioRxiv2018

Rationally-engineered reproductive barriers using CRISPR & CRISPRa: an evaluation of the synthetic species concept in Drosophila melanogaster

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.

synthetic biology↗

bioRxiv2017

Redkmer: An assembly-free pipeline for the identification of abundant and specific X-chromosome target sequences for X-shredding by CRISPR endonucleases

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 cant 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 An. gambiae, 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 An. gambiae, 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 sequences. 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 characterization of other biologically relevant sex chromosome sequences, a task that is frequently hampered by the repetitiveness of sex chromosome sequence content.

bioinformatics↗

bioRxiv2017

Cross-species Y chromosome function between malaria vectors of the Anopheles gambiae species complex

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, whether intentionally or contingent, between the major malaria vector species.

genetics↗

bioRxiv2016

Rapid evolution of female-biased genes among four species of Anopheles malaria mosquitoes

Understanding how phenotypic differences between males and females arise from the sex-biased expression of nearly identical genomes can often 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 vectors 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 suggests 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 only mate 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 reveals 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 demasculinizes the X in male reproductive tissues.

genomics↗