Site-specific genetic engineering of the Anopheles gambiae Y chromosome.

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

Improved CRISPR-based suppression gene drives mitigate resistance and impose a large reproductive load on laboratory-contained mosquito populations

Abstract CRISPR-based genes drives bias their own inheritance and can be used to modify entire populations of insect vectors of disease as a novel form of sustainable disease control. Gene drives designed to interfere with female fertility can suppress populations of the mosquito vector of malaria, however laboratory demonstrations showed strong unintended fitness costs and high levels of resistant mutations that limited the potential of the first generation of gene drives to spread. We describe three new gene drives designed to restrict spatio-temporal nuclease expression by using novel regulatory sequences. Two of the three new designs dramatically improve fitness and mitigate the creation and selection of resistance. We dissect the relative contributions of germline CRISPR activity versus embryonic CRISPR activity resulting from parental deposition, showing that the improved performance of the new designs is due to tighter germline restriction of the nuclease activity and significantly lower rates of end-joining repair in the embryo. Moreover, we demonstrate in laboratory-contained population experiments that these gene drives show remarkably improved invasion dynamics compared to the first generation drives, resulting in greater than 90% suppression of the reproductive output and a delay in the emergence of target site resistance, even at a loosely constrained target sequence. These results illustrate important considerations for gene drive design and will help expedite the development of gene drives designed to control malaria transmission in Africa

A synthetic sex ratio distortion system for the control of the human malaria mosquito.

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

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 F1 × F0 crossing scheme to overcome a severe bottleneck of male hybrid incompatibilities that enabled us to experimentally purify a genetically labeled A. gambiae Y chromosome in an A. arabiensis background. Whole genome sequencing (WGS) 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 1.85 MYA 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

Author Correction: A male-biased sex-distorter gene drive for the human malaria vector Anopheles gambiae.

An amendment to this paper has been published and can be accessed via a link at the top of the paper

Two decades of endemic dengue in Bangladesh (2000–2022): trends, seasonality, and impact of temperature and rainfall patterns on transmission dynamics

The objectives of this study were to compare dengue virus (DENV) cases, deaths, case-fatality ratio [CFR], and meteorological parameters between the first and the recent decades of this century (2000–2010 vs. 2011–2022) and to describe the trends, seasonality, and impact of change of temperature and rainfall patterns on transmission dynamics of dengue in Bangladesh. For the period 2000–2022, dengue cases and death data from Bangladesh’s Ministry of Health and Family Welfare’s website, and meteorological data from the Bangladesh Meteorological Department were analyzed. A Poisson regression model was performed to identify the impact of meteorological parameters on the monthly dengue cases. A forecast of dengue cases was performed using an autoregressive integrated moving average model. Over the past 23 yr, a total of 244,246 dengue cases were reported including 849 deaths (CFR = 0.35%). The mean annual number of dengue cases increased 8 times during the second decade, with 2,216 cases during 2000–2010 vs. 18,321 cases during 2011–2022. The mean annual number of deaths doubled (21 vs. 46), but the overall CFR has decreased by one-third (0.69% vs. 0.23%). Concurrently, the annual mean temperature increased by 0.49 °C, and rainfall decreased by 314 mm with altered precipitation seasonality. Monthly mean temperature (Incidence risk ratio [IRR]: 1.26), first-lagged rainfall (IRR: 1.08), and second-lagged rainfall (IRR: 1.17) were significantly associated with monthly dengue cases. The increased local temperature and changes in rainfall seasonality might have contributed to the increased dengue cases in Bangladesh

Radical remodeling of the Y chromosome in a recent radiation of malaria mosquitoes

open28openHall A.B.; Papathanos P.-A.; Sharma A.; Cheng C.; Akbari O.S.; Assour L.; Bergman N.H.; Cagnetti A.; Crisanti A.; Dottorini T.; Fiorentini E.; Galizi R.; Hnath J.; Jiang X.; Koren S.; Nolan T.; Radune D.; Sharakhova M.V.; Steele A.; Timoshevskiy V.A.; Windbichler N.; Zhang S.; Hahn M.W.; Phillippy A.M.; Emrich S.J.; Sharakhov I.V.; Tu Z.J.; Besansky N.J.Hall, A. B.; Papathanos, P. -A.; SHARMA DHAKAL, Apsara; Cheng, C.; Akbari, O. S.; Assour, L.; Bergman, N. H.; Cagnetti, A.; Crisanti, A.; Dottorini, T.; Fiorentini, E.; Galizi, R.; Hnath, J.; Jiang, X.; Koren, S.; Nolan, T.; Radune, D.; Sharakhova, M. V.; Steele, A.; Timoshevskiy, V. A.; Windbichler, N.; Zhang, Shangu; Hahn, M. W.; Phillippy, A. M.; Emrich, S. J.; Sharakhov, I. V.; Tu, Z. J.; Besansky, N. J

A CRISPR-Cas9 gene drive system targeting female reproduction in the malaria mosquito vector Anopheles gambiae.

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