Eliminating malaria vectors with precision-guided sterile males

Proceedings of the National Academy of Sciences, Год журнала: 2024, Номер 121(27)

Опубликована: Июнь 25, 2024

Controlling the principal African malaria vector, mosquito Anopheles gambiae , is considered essential to curtail transmission. However, existing vector control technologies rely on insecticides, which are becoming increasingly ineffective. Sterile insect technique (SIT) a powerful suppression approach that has successfully eradicated number of pests, yet A. toolkit lacks requisite for its implementation. SIT relies iterative mass releases nonbiting, nondriving, sterile males seek out and mate with monandrous wild females. Once mated, females permanently sterilized due mating-induced refractoriness, results in population subsequent generation. sterilization by traditional methods renders unfit, making creation precise genetic imperative. Here, we introduce technology termed precision-guided (pgSIT), inducible, programmed male female elimination wide-scale use campaigns. Using binary CRISPR strategy, cross separate engineered Cas9 gRNA strains disrupt male-fertility female-essential genes, yielding >99.5% sterility >99.9% lethality hybrid progeny. We demonstrate these genetically have good longevity, able induce sustained cage trials, predicted eliminate populations using mathematical models, them ideal candidates release. This work provides valuable addition biocontrol toolkit, enabling scalable SIT-like confinable, species-specific, safe species.

Язык: Английский

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Advances in the Epidemiology, Pathogenesis, Diagnostic Methods, and Vaccine Development of Dengue Fever: A Comprehensive Review

Viral Immunology, Год журнала: 2025, Номер unknown

Опубликована: Фев. 25, 2025

Dengue fever (DF) is a common mosquito-borne viral infection caused by any of the four dengue virus (DENV) serotypes. In recent years, global incidence DF has risen rapidly, which widely threatened health millions people in United States, Southeast Asia, and Western Pacific. The challenges for prevention control DENV have become increasingly severe. Over advances area research been continuously updating. this review, we provide an updated more in-depth overview epidemiology pathogenesis, along with progress diagnostic approaches (including methods to address cross-reactivity other flaviviruses) expanded discussion current vaccine development, such as CYD-TDV (Dengvaxia), TV003/TV005, new TAK-003. This comprehensive perspective aims offer references prevention, clinical diagnosis, disease.

Язык: Английский

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Loss-of-function in testis-specific serine/threonine protein kinase triggers male infertility in an invasive moth

Communications Biology, Год журнала: 2024, Номер 7(1)

Опубликована: Окт. 3, 2024

Genetic biocontrol technologies present promising and eco-friendly strategies for the management of pest insect-transmitted diseases. Although considerable advancements achieve in gene drive applications targeting mosquitoes, endeavors to combat agricultural pests have been somewhat restricted. Here, we identify that testis-specific serine/threonine kinases (TSSKs) family is uniquely expressed testes Cydia pomonella, a prominent global invasive species. We further generated male moths with disrupted expression TSSKs those using RNA interference CRISPR/Cas9 genetic editing techniques, resulting significant disruptions spermiogenesis, decreased sperm motility, hindered development eggs. Further explorations into underlying post-transcriptional regulatory mechanisms reveales involvement lnc117962 as competing endogenous (ceRNA) miR-3960, thereby regulating TSSKs. Notably, orchard trials demonstrates release strains can effectively suppress population growth. Our findings indicate could serve feasible avenue managing C. pomonella populations, offering insights potential controlling through sterile insect technique (gSIT) technology.

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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Advances and challenges in synthetic biology for mosquito control

Trends in Parasitology, Год журнала: 2023, Номер 40(1), С. 75 - 88

Опубликована: Ноя. 24, 2023

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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Differences in male Aedes aegypti and Aedes albopictus hearing systems facilitate recognition of conspecific female flight tones

iScience, Год журнала: 2024, Номер 27(7), С. 110264 - 110264

Опубликована: Июнь 13, 2024

When

Язык: Английский

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Activation of the G protein‐coupled sulfakinin receptor inhibits blood meal intake in the mosquito Aedes aegypti

The FASEB Journal, Год журнала: 2024, Номер 38(15)

Опубликована: Авг. 7, 2024

Abstract Little is known about the blood‐feeding physiology of arbovirus vector Aedes aegypti although this type mosquito to transmit infectious diseases dengue, Zika, yellow fever, and chikungunya. Blood feeding in female A. essential for egg maturation transmission disease agents between human subjects. Here, we identify sulfakinin receptor gene SKR from genome show that expressed at different developmental stages varied anatomical localizations adult (at three days after eclosion), with particularly high expression CNS. Knockingdown results increased blood meal intake, but microinjection thorax peptide 1 2 both inhibits dose dependently intake (and delays time course intake), which reversible antagonist. Sulfakinin ectopically mammalian cells CHO‐K1 responds stimulation persistent calcium spikes, blockable These data together suggest activation Gq protein‐coupled (i.e., calcium‐mobilizing) mosquitoes could serve as a strategic node future control reproduction/population transmission.

Язык: Английский

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Eliminating Malaria Vectors with Precision Guided Sterile Males

bioRxiv (Cold Spring Harbor Laboratory), Год журнала: 2023, Номер unknown

Опубликована: Июль 21, 2023

Controlling the principal African malaria vector, mosquito

Язык: Английский

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Precision-guided tools for malaria control

Proceedings of the National Academy of Sciences, Год журнала: 2024, Номер 121(32)

Опубликована: Июль 29, 2024

Язык: Английский

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