Converting endogenous genes of the malaria mosquito into simple non-autonomous gene drives for population replacement.
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ABSTRACT:
SUBMITTER: Hoermann A
PROVIDER: S-EPMC8043746 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description: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.
| S-EPMC6361204 | biostudies-literature
Project description:BackgroundThe siRNA and piRNA pathways have been shown in insects to be essential for regulation of gene expression and defence against exogenous and endogenous genetic elements (viruses and transposable elements). The vast majority of endogenous small RNAs produced by the siRNA and piRNA pathways originate from repetitive or transposable elements (TE). In D. melanogaster, TE-derived endogenous siRNAs and piRNAs are involved in genome surveillance and maintenance of genome integrity. In the medically relevant malaria mosquito Anopheles gambiae TEs constitute 12-16% of the genome size. Genetic variations induced by TE activities are known to shape the genome landscape and to alter the fitness in An. gambiae.ResultsHere, using bioinformatics approaches we analyzed the small RNA data sets from 6 libraries formally reported in a previous study and examined the expression of the mixed germline/somatic siRNAs and piRNAs produced in adult An. gambiae females. We characterized a large population of TE-derived endogenous siRNAs and piRNAs, which constitutes 56-60% of the total siRNA and piRNA reads in the analysed libraries. Moreover, we identified a number of protein coding genes producing gene-specific siRNAs and piRNAs that were generally expressed at much lower levels than the TE-associated small RNAs. Detailed sequence analysis revealed that An. gambiae piRNAs were produced by both "ping-pong" dependent (TE-associated piRNAs) and independent mechanisms (genic piRNAs). Similarly to D. melanogaster, more than 90% of the detected piRNAs were produced from TE-associated clusters in An. gambiae. We also found that biotic stress as blood feeding and infection with Plasmodium parasite, the etiological agent of malaria, modulated the expression levels of the endogenous siRNAs and piRNAs in An. gambiae.ConclusionsWe identified a large and diverse set of the endogenously derived siRNAs and piRNAs that share common and distinct aspects of small RNA expression across insect species, and inferred their impact on TE and gene activity in An. gambiae. The TE-specific small RNAs produced by both the siRNA and piRNA pathways represent an important aspect of genome stability and genetic variation, which might have a strong impact on the evolution of the genome and vector competence in the malaria mosquitoes.
| S-EPMC4423592 | biostudies-literature
Project description:Malaria is one of the deadliest vector-borne diseases in the world. Researchers are developing new genetic and conventional vector control strategies to attempt to limit its burden. Novel control strategies require detailed safety assessment to ensure responsible and successful deployments. Anopheles gambiae sensu stricto (s.s.) and Anopheles coluzzii, two closely related subspecies within the species complex Anopheles gambiae sensu lato (s.l.), are among the dominant malaria vectors in sub-Saharan Africa. These two subspecies readily hybridise and compete in the wild and are also known to have distinct niches, each with spatially and temporally varying carrying capacities driven by precipitation and land use factors. We model the spread and persistence of a population-modifying gene drive system in these subspecies across sub-Saharan Africa by simulating introductions of genetically modified mosquitoes across the African mainland and its offshore islands. We explore transmission of the gene drive between the two subspecies that arise from different hybridisation mechanisms, the effects of both local dispersal and potential wind-aided migration to the spread, and the development of resistance to the gene drive. Given the best current available knowledge on the subspecies' life histories, we find that an introduced gene drive system with typical characteristics can plausibly spread from even distant offshore islands to the African mainland with the aid of wind-driven migration, with resistance beginning to take over within a decade. Our model accounts for regional to continental scale mechanisms, and demonstrates a range of realistic dynamics including the effect of prevailing wind on spread and spatio-temporally varying carrying capacities for subspecies. As a result, it is well-placed to answer future questions relating to mosquito gene drives as important life history parameters become better understood.
| S-EPMC9191746 | biostudies-literature
Project description:Small laboratory cage trials of non-drive and gene-drive strains of the Asian malaria vector mosquito, Anopheles stephensi, were used to investigate release ratios and other strain properties for their impact on transgene spread during simulated population modification. We evaluated the effects of transgenes on survival, male contributions to next-generation populations, female reproductive success and the impact of accumulation of gene drive-resistant genomic target sites resulting from nonhomologous end-joining (NHEJ) mutagenesis during Cas9, guide RNA-mediated cleavage. Experiments with a non-drive, autosomally-linked malaria-resistance gene cassette showed 'full introduction' (100% of the insects have at least one copy of the transgene) within 8 weeks (? 3 generations) following weekly releases of 10:1 transgenic:wild-type males in an overlapping generation trial design. Male release ratios of 1:1 resulted in cages where mosquitoes with at least one copy of the transgene fluctuated around 50%. In comparison, two of three cages in which the malaria-resistance genes were linked to a gene-drive system in an overlapping generation, single 1:1 release reached full introduction in 6-8 generations with a third cage at ~80% within the same time. Release ratios of 0.1:1 failed to establish the transgenes. A non-overlapping generation, single-release trial of the same gene-drive strain resulted in two of three cages reaching 100% introduction within 6-12 generations following a 1:1 transgenic:wild-type male release. Two of three cages with 0.33:1 transgenic:wild-type male single releases achieved full introduction in 13-16 generations. All populations exhibiting full introduction went extinct within three generations due to a significant load on females having disruptions of both copies of the target gene, kynurenine hydroxylase. While repeated releases of high-ratio (10:1) non-drive constructs could achieve full introduction, results from the 1:1 release ratios across all experimental designs favor the use of gene drive, both for efficiency and anticipated cost of the control programs.
| S-EPMC6922335 | biostudies-literature
Project description:Inter-relationships among mosquito vectors, Plasmodium parasites, human ecology, and biotic and abiotic factors, drive malaria risk. Specifically, rural landscapes shaped by human activities have a great potential to increase the abundance of malaria vectors, putting many vulnerable people at risk. Understanding at which point the abundance of vectors increases in the landscape can help to design policies and interventions for effective and sustainable control. Using a dataset of adult female mosquitoes collected at 79 sites in malaria endemic areas in the Brazilian Amazon, this study aimed to (1) verify the association among forest cover percentage (PLAND), forest edge density (ED), and variation in mosquito diversity; and to (2) test the hypothesis of an association between landscape structure (i.e., PLAND and ED) and Nyssorhynchus darlingi (Root) dominance. Mosquito collections were performed employing human landing catch (HLC) (peridomestic habitat) and Shannon trap combined with HLC (forest fringe habitat). Nyssorhynchus darlingi abundance was used as the response variable in a generalized linear mixed model, and the Shannon diversity index (H') of the Culicidae community, PLAND, and the distance house-water drainage were used as predictors. Three ED categories were also used as random effects. A path analysis was used to understand comparative strengths of direct and indirect relationships among Amazon vegetation classes, Culicidae community, and Ny. darlingi abundance. Our results demonstrate that Ny. darlingi is negatively affected by H´ and PLAND of peridomestic habitat, and that increasing these variables (one-unit value at β0 = 768) leads to a decrease of 226 (P < 0.001) and 533 (P = 0.003) individuals, respectively. At the forest fringe, a similar result was found for H' (β1 = -218; P < 0.001) and PLAND (β1 = -337; P = 0.04). Anthropogenic changes in the Amazon vegetation classes decreased mosquito biodiversity, leading to increased Ny. darlingi abundance. Changes in landscape structure, specifically decreases in PLAND and increases in ED, led to Ny. darlingi becoming the dominant species, increasing malaria risk. Ecological mechanisms involving changes in landscape and mosquito species composition can help to understand changes in the epidemiology of malaria.
| S-EPMC7808592 | biostudies-literature
Project description:CRISPR-Cas9 nuclease-based gene drives have been developed toward the aim of control of the human malaria vector Anopheles gambiae Gene drives are based on an active source of Cas9 nuclease in the germline that promotes super-Mendelian inheritance of the transgene by homology-directed repair ("homing"). Understanding whether CRISPR-induced off-target mutations are generated in Anopheles mosquitoes is an important aspect of risk assessment before any potential field release of this technology. We compared the frequencies and the propensity of off-target events to occur in four different gene-drive strains, including a deliberately promiscuous set-up, using a nongermline restricted promoter for SpCas9 and a guide RNA with many closely related sites (two or more mismatches) across the mosquito genome. Under this scenario we observed off-target mutations at frequencies no greater than 1.42%. We witnessed no evidence that CRISPR-induced off-target mutations were able to accumulate (or drive) in a mosquito population, despite multiple generations' exposure to the CRISPR-Cas9 nuclease construct. Furthermore, judicious design of the guide RNA used for homing of the CRISPR construct, combined with tight temporal constriction of Cas9 expression to the germline, rendered off-target mutations undetectable. The findings of this study represent an important milestone for the understanding and managing of CRISPR-Cas9 specificity in mosquitoes, and demonstrates that CRISPR off-target editing in the context of a mosquito gene drive can be reduced to minimal levels.
| S-EPMC8179207 | biostudies-literature
Project description:A hallmark of neurodegenerative disease is focal onset of pathological protein aggregation, followed by progressive spread of pathology to connected brain regions. In amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), pathology is often associated with aggregation of TAR DNA-binding protein 43 (TDP-43). Although aggregated TDP-43 protein moves between cells, it is not clear whether and how this movement propagates the degeneration. Here, we have established a Drosophila model of human TDP-43 in which we initiated toxic expression of human TDP-43 focally within small groups of glial cells. We found that this focal onset kills adjacent neurons. Surprisingly, we show that this spreading death is caused by an endogenous retrovirus within the glia, which leads to DNA damage and death in adjacent neurons. These findings suggest a possible mechanism by which human retroviruses such as HERV-K might contribute to TDP-43-mediated propagation of neurodegeneration.
| S-EPMC6783360 | biostudies-literature
Project description:As pathogens that circumvent the host immune response are favoured by selection, so are host alleles that reduce parasite load. Such evolutionary processes leave their signature on the genes involved. Deciphering modes of selection operating on immune genes might reveal the nature of host-pathogen interactions and factors that govern susceptibility in host populations. Such understanding would have important public health implications.We analyzed polymorphisms in four mosquito immune genes (SP14D1, GNBP, defensin, and gambicin) to decipher selection effects, presumably mediated by pathogens. Using samples of Anopheles arabiensis, An. quadriannulatus and four An. gambiae populations, as well as published sequences from other Culicidae, we contrasted patterns of polymorphisms between different functional units of the same gene within and between populations. Our results revealed selection signatures operating on different time scales. At the most recent time scale, within-population diversity revealed purifying selection. Between populations and between species variation revealed reduced differentiation (GNBP and gambicin) at coding vs. noncoding- regions, consistent with balancing selection. McDonald-Kreitman tests between An. quadriannulatus and both sibling species revealed higher fixation rate of synonymous than nonsynonymous substitutions (GNBP) in accordance with frequency dependent balancing selection. At the longest time scale (>100 my), PAML analysis using distant Culicid taxa revealed positive selection at one codon in gambicin. Patterns of genetic variation were independent of exposure to human pathogens.Purifying selection is the most common form of selection operating on immune genes as it was detected on a contemporary time scale on all genes. Selection for "hypervariability" was not detected, but negative balancing selection, detected at a recent evolutionary time scale between sibling species may be rather common. Detection of positive selection at the deepest evolutionary time scale suggests that it occurs infrequently, possibly in association with speciation events. Our results provided no evidence to support the hypothesis that selection was mediated by pathogens that are transmitted to humans.
| S-EPMC2642720 | biostudies-literature
Project description:BACKGROUND:The Asian malaria mosquito, Anopheles stephensi, is a major urban malaria vector in the Middle East and on the Indian subcontinent. Early zygotic transcription, which marks the maternal-to-zygotic transition, has not been systematically studied in An. stephensi or any other Anopheles mosquitoes. Improved understanding of early embryonic gene expression in An. stephensi will facilitate genetic and evolutionary studies and help with the development of novel control strategies for this important disease vector. RESULTS:We obtained RNA-seq data in biological triplicates from four early An. stephensi embryonic time points. Using these data, we identified 70 and 153 pure early zygotic genes (pEZGs) under stringent and relaxed conditions, respectively. We show that these pEZGs are enriched in functional groups related to DNA-binding transcription regulators, cell cycle modulators, proteases, transport, and cellular metabolism. On average these pEZGs are shorter and have less introns than other An. stephensi genes. Some of the pEZGs may arise de novo while others have clear non-pEZG paralogs. There is no or very limited overlap between An. stephensi pEZGs and Drosophila melanogaster or Aedes aegypti pEZGs. Interestingly, the upstream region of An. stephensi pEZGs lack significant enrichment of a previously reported TAGteam/VBRGGTA motif found in the regulatory region of pEZGs in D. melanogaster and Ae. aegypti. However, a GT-rich motif was found in An. stephensi pEZGs instead. CONCLUSIONS:We have identified a number of pEZGs whose predicted functions and structures are consistent with their collective roles in the degradation of maternally deposited components, activation of the zygotic genome, cell division, and metabolism. The pEZGs appear to rapidly turn over within the Dipteran order and even within the Culicidae family. These pEZGs, and the shared regulatory motif, could provide the promoter or regulatory sequences to drive gene expression in the syncytial or early cellular blastoderm, a period when the developing embryo is accessible to genetic manipulation. In addition, these molecular resources may be used to achieve sex separation of mosquitoes for sterile insect technique.
| S-EPMC6304767 | biostudies-literature
Project description:Physical and genetic maps have been used for chromosomal localization of genes in vectors of infectious diseases. The availability of polytene chromosomes in malaria mosquitoes provides a unique opportunity to precisely map genes of interest. We report the physical mapping of two actin genes on polytene chromosomes of the major malaria vector in the Amazon, Anopheles darlingi (Diptera: Culicidae). Clones with actin gene sequences were obtained from a cDNA library constructed from RNA isolated from adult females and males of An. darlingi. Each of the two clones was mapped to a unique site on chromosomal arm 2L in subdivisions 21A (clone pl05-A04) and 23B (clone pl17-G06). The obtained results, together with previous mapping data, provide a suitable basis for comparative genomics and for establishing chromosomal homologies among major malaria vectors.
| S-EPMC3477284 | biostudies-literature