Generating prophylactic immunity against arboviruses in vertebrates and invertebrates

Nature reviews. Immunology, Год журнала: 2024, Номер 24(9), С. 621 - 636

Опубликована: Апрель 3, 2024

Язык: Английский

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Aedes aegypti Controls Ae. aegypti: SIT and IIT—An Overview

Journal of the American Mosquito Control Association, Год журнала: 2024, Номер 40(1), С. 32 - 49

Опубликована: Март 1, 2024

ABSTRACT The sterile insect technique (SIT) and the incompatible (IIT) are emerging potentially revolutionary tools for controlling Aedes aegypti (L.), a prominent worldwide mosquito vector threat to humans that is notoriously difficult reduce or eliminate in intervention areas using traditional integrated management (IVM) approaches. Here we provide an overview of discovery, development, application SIT IIT Ae. control, innovations advances technology, including transgenics, could elevate these techniques sustainable solution when combined with other IVM practices.

Язык: Английский

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Targeting sex determination to suppress mosquito populations

eLife, Год журнала: 2024, Номер 12

Опубликована: Янв. 30, 2024

Each year, hundreds of millions people are infected with arboviruses such as dengue, yellow fever, chikungunya, and Zika, which all primarily spread by the notorious mosquito Aedes aegypti . Traditional control measures have proven insufficient, necessitating innovations. In response, here we generate a next-generation CRISPR-based precision-guided sterile insect technique (pgSIT) for Ae. that disrupts genes essential sex determination fertility, producing predominantly males can be deployed at any life stage. Using mathematical models empirical testing, demonstrate released pgSIT effectively compete with, suppress, eliminate caged populations. This versatile species-specific platform has potential field deployment to wild populations disease vectors.

Язык: Английский

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Regulatory and policy considerations for the implementation of gene drive-modified mosquitoes to prevent malaria transmission

Transgenic Research, Год журнала: 2023, Номер 32(1-2), С. 17 - 32

Опубликована: Март 15, 2023

Abstract Gene drive-modified mosquitoes (GDMMs) are being developed as possible new tools to prevent transmission of malaria and other mosquito-borne diseases. To date no GDMMs have yet undergone field testing. This early stage is an opportune time for developers, supporters, users begin consider the potential regulatory requirements eventual implementation these technologies in national or regional public health programs, especially some practical implications may take considerable planning, coordination address. Several currently unresolved questions pertinent examined, including: how product will be defined; what registration/approval process placing GDMM products on market; transboundary movement can addressed; role might played by existing multinational bodies agreements authorization decisions. Regulation policies applied registration genetically modified organisms living mosquito assessed relevance use case Africa. Multiple authorities likely involved decision-making, according laws place within each country certain classes. Requirements under Cartagena Protocol Biodiversity considered relevant most countries, frameworks conventional pesticide, medical, biocontrol products. Experience suggests that standard processes, evidence requirements, liability differ from country. Regional mechanisms useful address important challenges.

Язык: Английский

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Targeting sex determination to suppress mosquito populations

eLife, Год журнала: 2023, Номер 12

Опубликована: Сен. 27, 2023

Full text Figures and data Peer review Side by side Abstract eLife assessment Introduction Results Discussion Methods Data availability References Article author information Metrics Each year, hundreds of millions people are infected with arboviruses such as dengue, yellow fever, chikungunya, Zika, which all primarily spread the notorious mosquito Aedes aegypti. Traditional control measures have proven insufficient, necessitating innovations. In response, here we generate a next-generation CRISPR-based precision-guided sterile insect technique (pgSIT) for Ae. aegypti that disrupts genes essential sex determination fertility, producing predominantly males can be deployed at any life stage. Using mathematical models empirical testing, demonstrate released pgSIT effectively compete with, suppress, eliminate caged populations. This versatile species-specific platform has potential field deployment to wild populations disease vectors. valuable paper builds on method, previously conceptualized validated, genetic The called pgSIT, uses integrated CRISPR-Cas9 based constructs generate, in certain combinations genotypes, mutations cause both male sterility female inviability. Release genotypes sufficiently large numbers lead an inundation local population this localised suppression, represents effective method problematic convincing will interest anyone working vector strategies. https://doi.org/10.7554/eLife.90199.3.sa0 About assessments is principal several deadly viruses, including Zika. Unfortunately, its geographic been exacerbated climate change, global trade, preference anthropogenic habitats, 3.9 billion presently risk (Brady et al., 2012). Traditionally, achieved through insecticides, though developing resistance rendered them less effective. Moreover, insecticides harmful non-target species, pollinators, direct exposure dangerous humans pets (Hemingway 2002; Lu 2020; Sánchez-Bayo, 2021; Trdan, 2016). Alternative approaches include preventative designed reduce habitat reduction or larval predation, these also impractical introduce invasive species. Notwithstanding, continuous expansion species demonstrates current insufficient. biological developed specific Sterile Insect Technique (SIT), Wolbachia-mediated Incompatible (WIIT), Female-Specific Insects Carrying Dominant Lethal gene (fsRIDL), Inherited Female Elimination Genetically Encoded Nucleases Interrupt Alleles (IFEGENIA), Gene Drive (GD) SIT (Knipling, 1955; Laven, 1967; Spinner 2022; Smidler 2023; Li 2017; Kandul 2019b; 2021). Here, radiation-sterilized (♂'s), ♂'s harboring Wolbachia, repeatedly mass mate females (♀'s) typically only once, thereby reducing over time. fsRIDL genetically sex-sort ♂'s, eggs. IFEGENIA female-essential gene, femaleless (fle), demonstrating complete sexing via heritable daughter gynecide. GD leverage CRISPR precise DNA cutting repair into Two types drives engineered: suppression collapses population, whereas modification cargo potentially block chain transmission targeted (Champer 2016; Marshall 2019; Raban Akbari, Wang works simultaneously disrupting ♀ viability ♂ fertility during development sex-sorted viable sterilized ♂'s. Previously, showed ♀-specific flight muscle (fem-myo) induced flightless phenotype ♀'s (Li However, approach would still result transgenic being may desirable than removing altogether. To further build upon system, instead lethality, simplifying process generating release. Our herein-developed three aegypti: doublessex (dsx), intersex (ix), β-Tubulin 85D (βTub), induce (⚥) transformation, near-complete ♂/♀ sterility. We competitive multiple releases suppress cage Notably, modeling indicates technology wide range realistic performance parameters fitness costs. Overall, provides option release males, bringing significantly closer deployable Additionally, develop rapid diagnostic assay detect transgene presence could used alongside tests. Taken together, demonstrated target produce providing alternate system controlling vector. Disrupting lethality and/or ⚥ transformation aegypti, either dsx (AAEL009114) ix (AAEL010217) genes, associated differentiation insects (Chase Baker, 1995; Garrett-Engele Kyrou 2018; Mysore 2015; Salvemini 2011; Scali 2005; Trujillo-Rodríguez located chromosome 1R alternatively spliced two (dsxF1 dsxF2) one ♂-specific (dsxM) isoforms (Salvemini 2011). Exon 5b retained dsxF1 dsxF2 transcripts out dsxM transcript. Therefore, disrupt isoforms, selected four gRNAs exon (Supplementary file 1a) assembled pBac gRNAdsx plasmid (Figure 1A). 3L orthologous required sexual Drosophila melanogaster 1995). While D. does not undergo sex-specific alternative splicing expressed sexes, interacts dsxF, but dsxM, resulting activity (Garrett-Engele 2002). ix, engineered gRNAix cassette targeting 1 1A, Supplementary 1a). determine effects separate strains were generated assessed crossing Nup50-Cas9 (hereafter, Cas9) explore maternal Cas9 carryover, crosses conducted reciprocally 1B). Remarkably, disruption transformed trans-hemizygous intersexes (⚥'s), independent whether was inherited from mother (i.e., ♀Cas9), father ♂Cas9). These gRNAdsx/+; Cas9/+ exhibited malformed morphological features, mutant maxillary palps, abnormal genitalia, ovaries 1D, Figure 1—figure supplement 1, 1b). ~50% ⚥'s able blood feed, very few unhatchable similar fraction gRNAix/+; fed laid hatchable eggs 1C, 1c). Sanger sequencing confirmed expected loci 2). Importantly, did observe changes ear anatomy, wing beat frequency (WBF), phonotactic behaviors suggesting normal courtship behavior 3, findings reveal efficiently while requires disruption. 3 supplements see Download asset Open CRISPR/Cas9-mediated affects morphology fecundity. (A) Schematic map gRNA constructs. harbor 3xP3-tdTomato marker guiding female-specific doublesex (dsx) female-active (ix) respectively. (B) A schematic cross between homozygous hemizygous gRNAdsx/+ gRNAix/+ mosquitoes F1 (♀'s). Reciprocal established generated: gRNA/+; ♀Cas9/+ Cas9; ♂Cas9/+ paternal Cas9. Then, crossed wild-type (WT) their fecundity assessed. comparison ratio trans-hemizygous, those WT ♀'s. bar plot shows means standard deviation (± SD) triple replicates (n = 3). presentsone strain each construct, gRNAdsx#1 gRNAix#1, results (all found (D) All gRNAdsx#1/+; gRNAix#1/+; male-specific features (red arrows D), had reduced fecundity, (⚥'s). Statistical significance mean differences estimated using two-sided Student's t-test equal variance (ns: p ≥ 0.05, *p < **p 0.01, ***p 0.001). Source provided 1. Multiplexed guide CRISPR-mediated gRNAdsx,ix,βTub construct combines described above More specifically, harbors six gRNAs: 5b; 1; (βTub, AAEL019894) 2A, βTub specifically testes (Akbari 2013; Degner Gamez 2020) sperm maturation, elongation, motility (Chen Three measure survival, fitness, trans-hemizygous) progeny 2B, C, 2—figure 1). integration sites, performed genome gRNAdsx,ix,βTub#1 gRNAdsx,ix,βTub) harbored single copy inserted data, validated diverse intended sites RNA revealed sequences 4–6, 1j, k). copies mutagenesis targets simultaneously, desired phenotypic redundancy increased resilience. 2 8 nearly maps (box) construct. Red show relative locations (box). simultaneous dsx, genes. Violet Box 5a represent exons. reciprocal Cas9, marked Opie2-CFP, (aka. pgSIT) progeny. Relative positions (bar color corresponds color), insertions (Nup50-Cas9 strain1) indicated pairs chromosomes. assess mosquitoes, ♀'s, (C) Comparison ratio, gRNAdsx,ix,βTub#1, mosquitoes. ± (SD) 1d). Blood feeding assays (⚥'s): pgSIT♀Cas9 pgSIT♂Cas9 ⚥'s. efficiency, individual mated allowed feed anesthetized mouse inside smaller (24.5 × 24.5 cm) larger (60 60 cm), recorded time: (1) initiate time bite); (2) time). duration SD 30 30) background 1e). (E) Flight 24 hr vertical monitoring (DAM, 1f). (n=24). (F) Mating offspring produced (pgSIT♀Cas9) (pgSIT♂Cas9) 1h). biologically groups 50 3) experimental condition. incapacitated next tested βTub, resulted death most ⚥'s, no 5–6% survived adulthood 2C). Interestingly, around 95% failed eclose, (termed respectively, eclose more pronounced phenotypes single-gene 1) 2C, vast majority adult they 2D, 1e), fly frequently 2E, 1f), abdomens wings 1b), 4x-reduced longevity 8A, B, 1g). combination induces unfit mostly mating ability ~94% eclosed due surviving Both pgSIT♀cas9 completely Surprisingly, despite our assumption pgSIT♀cas9♂ roughly equivalent, developmental times slightly delayed 8D). Nevertheless, different 8, 1g), re-mating 2F, deposition some costs, however avoided source observed effects, likely capable inducing suppression. enough populations, multigenerational, experiments. At generation, mosquitos cages introduction frequencies (pgSIT:WT – 1:1, 5:1, 10:1, 20:1, 40:1; 3A 1i). behaved similarly, high-release ratios (20:1, 40:1) eliminated five generations 3B, C). With lower 10:1 significant rates hatched cages. No effect C confirm larvae screened transgenesis markers generation. cages, scored markers, few, indicating indeed gradually Population multigenerational Multiple (pgSIT):wild-type ratios, 40:1, triplicate. diagram depicts experiments 5:1 ratio. start first generation (G1) 250 mature similarly aged cage, virgin added. days before removed, collected. counted, 100 randomly seed (G2). emerged (N♂) (5N♂) N♀ added later. remaining hatching score markers. procedure repeated assessed: (pgSIT♀Cas9), (pgSIT♂Cas9). Multigeneration plotting normalized percentage (C). Points triplicate sample (n=3). Theoretical effectiveness non-optimal outcomes, simulated 10,000 adults 4). Weekly up 500 (♀ ♂) per 1–52 weeks. scale chosen considering common interventions (Carvalho 2015) >30 day temperate climates (Mordecai 2019). assume same survival probability wild-laid eggs; if do higher mortality, equivalent preliminary sensitivity analysis outcomes sensitive 4—figure considered elimination (the stochastic simulations elimination) window protection ≥50% ≥90% population) explored how vary number releases, size interval, rate, competitiveness, viability. Suppression schedule (number, size, interval releases), Model-predicted efficacy pgSIT-induced then detail function series depicted 4. exceeding years (a benchmark interruption transmission) generally 0 (complete inviability) 0.15, sterility) scheme exceeds ~23 ≥100 4A). Achieving places tighter restrictions safe ballpark 0–0.10, given ~26 4B). suggest product profile 0–0.10. falls well within bounds, ~0.01. 4 egg scheme, sterility, mixing MGDrivE simulation framework2 1l. identified model describing varied factorial assessing elimination), population). adult, weekly 52. Regions parameter space 90% purple, light blue. Time-series dynamics select sets a–h. total denoted red, Cas9-carrying blue shaded region protection. Imperfect inviability probabilities elimination; lasts 0–0.15 schemes. (x-axis), (y-axis), minimum achieve (shadings, key). set adult. square 0% 10% 10%. ~20–32 systems having parameters. Mitigation tools detection Being rapidly identify transgenes environment mixtures captured important applications biocontrol tools. PCR-based tests exist, difficult implement field. simple readout desired. future trials, adapted enzymatic nucleic acid sequence reporter (SENSR) (Brogan 2021) fragments SENSR, triggers activation Ruminococcus flavefaciens CasRx/gRNA collateral cleaves quenched probe, fluorescent Dalla Benetta Brogan 0:1; 1:1; 1:5; 1:10; 1:25; 1:50, template (NTC) challenge SENSR. SENSR detected even lowest dilution, signal WT-only NTC controls every sequence, reaching half-maximum fluorescence (HMF) 20min pools instrumental tool trials. robust stable addressing vectors, indicate elimination, reinforce finding. support trials other trangene-based technologies, identifying transgenes. frequencies. That said, limitations. First, determined 100% sterile, ~1% progeny, stem fewer previous saw it new additional ensure achieved. Even become fixed natural likelihood off-target becoming exceedingly low. mitigate negative impacts, employed CHOPCHOP V3.0.0 (https://chopchop.cbu.uib.no) selection gRNAs, minimize occurrence genomic cleavage events. Furthermore, releasing carried rounds. event undesired introduced eradicated subsequent rounds naturally Second, noticed when maternally, overabundance present (Kandul 2020). ensuring paternally

Язык: Английский

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Manipulating the Destiny of Wild Populations Using CRISPR

Annual Review of Genetics, Год журнала: 2023, Номер 57(1), С. 361 - 390

Опубликована: Сен. 19, 2023

Genetic biocontrol aims to suppress or modify populations of species protect public health, agriculture, and biodiversity. Advancements in genome engineering technologies have fueled a surge research this field, with one gene editing technology, CRISPR, leading the charge. This review focuses on current state CRISPR for genetic pests highlights progress ongoing challenges using these approaches.

Язык: Английский

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Efficient sex separation by exploiting differential alternative splicing of a dominant marker in Aedes aegypti

PLoS Genetics, Год журнала: 2023, Номер 19(11), С. e1011065 - e1011065

Опубликована: Ноя. 27, 2023

Only female mosquitoes consume blood giving them the opportunity to transmit deadly human pathogens. Therefore, it is critical remove females before conducting releases for genetic biocontrol interventions. Here we describe a robust sex-sorting approach termed SEPARATOR ( S exing E lement P roduced by A lternative R NA-splicing of T ransgenic O bservable eporter) that exploits sex-specific alternative splicing an innocuous reporter ensure exclusive dominant male-specific expression. Using SEPARATOR, demonstrate reliable sex selection from early larval and pupal stages in Aedes aegypti , use Complex Object Parametric Analyzer Sorter (COPAS) scalable high-throughput sex-selection first instar larvae. Additionally, this sequence transcriptomes males find several genes are sex-specifically expressed. can simplify mass production release programs designed be cross-species portable should instrumental

Язык: Английский

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Population suppression with dominant female-lethal alleles is boosted by homing gene drive

BMC Biology, Год журнала: 2024, Номер 22(1)

Опубликована: Сен. 11, 2024

Язык: Английский

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Improving the suppressive power of homing gene drive by co-targeting a distant-site female fertility gene

Nature Communications, Год журнала: 2024, Номер 15(1)

Опубликована: Окт. 26, 2024

Gene drive technology has the potential to address major biological challenges. Well-studied homing suppression drives have been shown be highly efficient in Anopheles mosquitoes, but for other organisms, lower rates of conversion prevent elimination target population. To tackle this issue, we propose a gene design that two targets: site where takes place, and distant cleavage induces population suppression. We model find two-target system allows occur over much wider range efficiency. Specifically, cutting efficiency now determines suppressive power drive, rather than as standard drives. construct Drosophila melanogaster show both components function successfully. However, embryo from maternal deposition well fitness costs female heterozygotes remain significant challenges Overall, our improved ease problems associated with many species is less efficient.

Язык: Английский

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Evaluation of ebony as a potential selectable marker for genetic sexing in Aedes aegypti

Parasites & Vectors, Год журнала: 2025, Номер 18(1)

Опубликована: Фев. 25, 2025

Abstract Background Aedes aegypti is expected to invade previously unoccupied areas, mainly due the climate change, increase in travel and trade activities continuous transformation of rural environment into urban areas. The sterile insect technique (SIT), which relies on mass production release males, an environmentally friendly approach that can be applied for population control Ae. aegypti. SIT programs greatly benefited by a genetic sexing strain (GSS) reliable sex sorting system minimize any accidental female release. Visually detectable or conditionally lethal selectable markers used development new GSSs. In this study, we evaluated suitability competence mutant ebony GSS. gene known involved pigmentation pathway several dipteran insects, including . Methods An knockout was developed though CRISPR/Cas9 mutagenesis. G 0 individuals with desired phenotype were crossed, progeny screened every generation. PCR sequencing performed using gDNA from pulled leg determine genotype. Quality tests, pupae adult recovery rates, male ratio fecundity, line whether mutation confers fitness cost. Results carrying 5-bp deletion obtained, presented darker head siphon phenotypes at larval stage. However, analysis revealed results incomplete penetrance variable expressivity. establishment pure not possible because costs conferred mutation. Conclusions adequacy as marker GSS assessed. Despite its clear early development, homozygous phenotypic inconsistency loss fertility. These drawbacks clearly indicate particular suitable Nonetheless, it cannot excluded different will lead expression profile viable line. Graphical

Язык: Английский

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