Regulating the expression of gene drives is key to increasing their invasive potential and the mitigation of resistance.

Homing-based gene drives use a germline source of nuclease to copy themselves at specific target sites in a genome and bias their inheritance. Such gene drives can be designed to spread and deliberately suppress populations of malaria mosquitoes by impairing female fertility. However, strong unintended fitness costs of the drive and a propensity to generate resistant mutations can limit a gene drive's potential to spread. Alternative germline regulatory sequences in the drive element confer improved fecundity of carrier individuals and reduced propensity for target site resistance. This is explained by reduced rates of end-joining repair of DNA breaks from parentally deposited nuclease in the embryo, which can produce heritable mutations that reduce gene drive penetrance. We tracked the generation and selection of resistant mutations over the course of a gene drive invasion of a population. Improved gene drives show faster invasion dynamics, increased suppressive effect and later onset of target site resistance. Our results show that regulation of nuclease expression is as important as the choice of target site when developing a robust homing-based gene drive for population suppression.

Resistance to a CRISPR-based gene drive at an evolutionarily conserved site is revealed by mimicking genotype fixation.

CRISPR-based homing gene drives can be designed to disrupt essential genes whilst biasing their own inheritance, leading to suppression of mosquito populations in the laboratory. This class of gene drives relies on CRISPR-Cas9 cleavage of a target sequence and copying ('homing') therein of the gene drive element from the homologous chromosome. However, target site mutations that are resistant to cleavage yet maintain the function of the essential gene are expected to be strongly selected for. Targeting functionally constrained regions where mutations are not easily tolerated should lower the probability of resistance. Evolutionary conservation at the sequence level is often a reliable indicator of functional constraint, though the actual level of underlying constraint between one conserved sequence and another can vary widely. Here we generated a novel adult lethal gene drive (ALGD) in the malaria vector Anopheles gambiae, targeting an ultra-conserved target site in a haplosufficient essential gene (AGAP029113) required during mosquito development, which fulfils many of the criteria for the target of a population suppression gene drive. We then designed a selection regime to experimentally assess the likelihood of generation and subsequent selection of gene drive resistant mutations at its target site. We simulated, in a caged population, a scenario where the gene drive was approaching fixation, where selection for resistance is expected to be strongest. Continuous sampling of the target locus revealed that a single, restorative, in-frame nucleotide substitution was selected. Our findings show that ultra-conservation alone need not be predictive of a site that is refractory to target site resistance. Our strategy to evaluate resistance in vivo could help to validate candidate gene drive targets for their resilience to resistance and help to improve predictions of the invasion dynamics of gene drives in field populations.

The creation and selection of mutations resistant to a gene drive over multiple generations in the malaria mosquito.

Gene drives have enormous potential for the control of insect populations of medical and agricultural relevance. By preferentially biasing their own inheritance, gene drives can rapidly introduce genetic traits even if these confer a negative fitness effect on the population. We have recently developed gene drives based on CRISPR nuclease constructs that are designed to disrupt key genes essential for female fertility in the malaria mosquito. The construct copies itself and the associated genetic disruption from one homologous chromosome to another during gamete formation, a process called homing that ensures the majority of offspring inherit the drive. Such drives have the potential to cause long-lasting, sustainable population suppression, though they are also expected to impose a large selection pressure for resistance in the mosquito. One of these population suppression gene drives showed rapid invasion of a caged population over 4 generations, establishing proof of principle for this technology. In order to assess the potential for the emergence of resistance to the gene drive in this population we allowed it to run for 25 generations and monitored the frequency of the gene drive over time. Following the initial increase of the gene drive we observed a gradual decrease in its frequency that was accompanied by the spread of small, nuclease-induced mutations at the target gene that are resistant to further cleavage and restore its functionality. Such mutations showed rates of increase consistent with positive selection in the face of the gene drive. Our findings represent the first documented example of selection for resistance to a synthetic gene drive and lead to important design recommendations and considerations in order to mitigate for resistance in future gene drive applications.

Crystallographic analyses illustrate significant plasticity and efficient recoding of meganuclease target specificity.

The retargeting of protein-DNA specificity, outside of extremely modular DNA binding proteins such as TAL effectors, has generally proved to be quite challenging. Here, we describe structural analyses of five different extensively retargeted variants of a single homing endonuclease, that have been shown to function efficiently in ex vivo and in vivo applications. The redesigned proteins harbor mutations at up to 53 residues (18%) of their amino acid sequence, primarily distributed across the DNA binding surface, making them among the most significantly reengineered ligand-binding proteins to date. Specificity is derived from the combined contributions of DNA-contacting residues and of neighboring residues that influence local structural organization. Changes in specificity are facilitated by the ability of all those residues to readily exchange both form and function. The fidelity of recognition is not precisely correlated with the fraction or total number of residues in the protein-DNA interface that are actually involved in DNA contacts, including directional hydrogen bonds. The plasticity of the DNA-recognition surface of this protein, which allows substantial retargeting of recognition specificity without requiring significant alteration of the surrounding protein architecture, reflects the ability of the corresponding genetic elements to maintain mobility and persistence in the face of genetic drift within potential host target sites.

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

Y chromosomes control essential male functions in many species, including sex determination and fertility. However, because of obstacles posed by repeat-rich heterochromatin, knowledge of Y chromosome sequences is limited to a handful of model organisms, constraining our understanding of Y biology across the tree of life. Here, we leverage long single-molecule sequencing to determine the content and structure of the nonrecombining Y chromosome of the primary African malaria mosquito, Anopheles gambiae We find that the An. gambiae Y consists almost entirely of a few massively amplified, tandemly arrayed repeats, some of which can recombine with similar repeats on the X chromosome. Sex-specific genome resequencing in a recent species radiation, the An. gambiae complex, revealed rapid sequence turnover within An. gambiae and among species. Exploiting 52 sex-specific An. gambiae RNA-Seq datasets representing all developmental stages, we identified a small repertoire of Y-linked genes that lack X gametologs and are not Y-linked in any other species except An. gambiae, with the notable exception of YG2, a candidate male-determining gene. YG2 is the only gene conserved and exclusive to the Y in all species examined, yet sequence similarity to YG2 is not detectable in the genome of a more distant mosquito relative, suggesting rapid evolution of Y chromosome genes in this highly dynamic genus of malaria vectors. The extensive characterization of the An. gambiae Y provides a long-awaited foundation for studying male mosquito biology, and will inform novel mosquito control strategies based on the manipulation of Y chromosomes.

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.

Expression of the glycolytic enzymes enolase and lactate dehydrogenase during the early phase of Toxoplasma differentiation is regulated by an intron retention mechanism.

The intracellular parasite Toxoplasma gondii converts from a rapidly replicating tachyzoite form during acute infection to a quiescent encysted bradyzoite stage that persists inside long-lived cells during chronic infection. Bradyzoites adopt reduced metabolism and slow replication while waiting for an opportunity to recrudesce the infection within the host. Interconversion between these two developmental stages is characterized by expression of glycolytic isoenzymes that play key roles in parasite metabolism. The parasite genome encodes two isoforms of lactate dehydrogenase (LDH1 and LDH2) and enolase (ENO1 and ENO2) that are expressed in a stage-specific manner. Expression of different isoforms of these enzymes allows T. gondii to rapidly adapt to diverse metabolic requirements necessary for either a rapid replication of the tachyzoite stage or a quiescent lifestyle typical of the bradyzoites. Herein we identified unspliced forms of LDH and ENO transcripts produced during transition between these two parasite stages suggestive of an intron retention mechanism to promptly exchange glycolytic isoforms for rapid adaptation to environmental changes. We also identified key regulatory elements in the ENO transcription units, revealing cooperation between the ENO2 5'-untranslated region and the ENO2 intron, along with identifying a role for the ENO1 3'-untranslated region in stage-specific expression.

The germline of the malaria mosquito produces abundant miRNAs, endo-siRNAs, piRNAs and 29-nt small RNAs.

BACKGROUND: Small RNAs include different classes essential for endogenous gene regulation and cellular defence against genomic parasites. However, a comprehensive analysis of the small RNA pathways in the germline of the mosquito Anopheles gambiae has never been performed despite their potential relevance to reproductive capacity in this malaria vector. RESULTS: We performed small RNA deep sequencing during larval and adult gonadogenesis and find that they predominantly express four classes of regulatory small RNAs. We identified 45 novel miRNA precursors some of which were sex-biased and gonad-enriched , nearly doubling the number of previously known miRNA loci. We also determine multiple genomic clusters of 24-30 nt Piwi-interacting RNAs (piRNAs) that map to transposable elements (TEs) and 3'UTR of protein coding genes. Unusually, many TEs and the 3'UTR of some endogenous genes produce an abundant peak of 29-nt small RNAs with piRNA-like characteristics. Moreover, both sense and antisense piRNAs from TEs in both Anopheles gambiae and Drosophila melanogaster reveal novel features of piRNA sequence bias. We also discovered endogenous small interfering RNAs (endo-siRNAs) that map to overlapping transcripts and TEs. CONCLUSIONS: This is the first description of the germline miRNome in a mosquito species and should prove a valuable resource for understanding gene regulation that underlies gametogenesis and reproductive capacity. We also provide the first evidence of a piRNA pathway that is active against transposons in the germline and our findings suggest novel piRNA sequence bias. The contribution of small RNA pathways to germline TE regulation and genome defence in general is an important finding for approaches aimed at manipulating mosquito populations through the use of selfish genetic elements.

Genetic Diversity and Population Structure of the Invasive Oriental Fruit Fly, Bactrocera dorsalis (Diptera: Tephritidae) in Burkina Faso.

Bactrocera dorsalis Hendel is a highly invasive horticultural pest that is of major economic importance worldwide. In Burkina Faso, it is one of the main insect pests that affects the production and exportation of mangos. Understanding the biology and the genetic dynamics of this insect pest provides crucial information for the development of effective control measures. The aim of this study was to understand the distribution, diversity, and genetic structure of B. dorsalis in Burkina Faso. Male flies were collected transversally in Burkina Faso and analyzed by PCR using 10 microsatellite markers. The results showed an abundance of B. dorsalis varying from 87 to 2986 flies per trap per day at the different sampling sites. The genetic diversity was high at all sites, with an average Shannon's Information Index (I) of 0.72 per site. The gene flow was high between study populations and ranged from 10.62 to 27.53 migrants. Bayesian admixture analysis showed no evidence of structure, while Discriminant Analysis of Principal Components identified three weakly separated clusters in the population of B. dorsalis in Burkina Faso. The results of this study could be used to optimize the effectiveness of current control interventions and to guide the implementation of new, innovative, and sustainable strategies.

Anti-CRISPR Anopheles mosquitoes inhibit gene drive spread under challenging behavioural conditions in large cages.

CRISPR-based gene drives have the potential to spread within populations and are considered as promising vector control tools. A doublesex-targeting gene drive was able to suppress laboratory Anopheles mosquito populations in small and large cages, and it is considered for field application. Challenges related to the field-use of gene drives and the evolving regulatory framework suggest that systems able to modulate or revert the action of gene drives, could be part of post-release risk-mitigation plans. In this study, we challenge an AcrIIA4-based anti-drive to inhibit gene drive spread in age-structured Anopheles gambiae population under complex feeding and behavioural conditions. A stochastic model predicts the experimentally-observed genotype dynamics in age-structured populations in medium-sized cages and highlights the necessity of large-sized cage trials. These experiments and experimental-modelling framework demonstrate the effectiveness of the anti-drive in different scenarios, providing further corroboration for its use in controlling the spread of gene drive in Anopheles.

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.

qSanger: Quantification of Genetic Variants in Bacterial Cultures by Sanger Sequencing.

Genetic variations such as mutations and recombinations arise spontaneously in all cultured organisms. Although it is possible to identify nonneutral mutations by selection or counterselection, the identification of neutral mutations in a heterogeneous population usually requires expensive and time-consuming methods such as quantitative or droplet polymerase chain reaction and high-throughput sequencing. Neutral mutations could even become dominant under changing environmental conditions enforcing transitory selection or counterselection. We propose a novel method, which we called qSanger, to quantify DNA using amplitude ratios of aligned electropherogram peaks from mixed Sanger sequencing reads. Plasmids expressing enhanced green fluorescent protein and mCherry fluorescent markers were used to validate qSanger both in vitro and in cotransformed Escherichia coli via quantitative polymerase chain reaction and fluorescence quantifications. We show that qSanger allows the quantification of genetic variants, including single-base natural polymorphisms or de novo mutations, from mixed Sanger sequencing reads, with substantial reduction of labor and costs compared to canonical approaches.

Single-cell profiling of Anopheles gambiae spermatogenesis defines the onset of meiotic silencing and premeiotic overexpression of the X chromosome.

Understanding development and genetic regulation in the Anopheles gambiae germline is essential to engineer effective genetic control strategies targeting this malaria mosquito vector. These include targeting the germline to induce sterility or using regulatory sequences to drive transgene expression for applications such as gene drive. However, only very few germline-specific regulatory elements have been characterised with the majority showing leaky expression. This has been shown to considerably reduce the efficiency of current genetic control strategies, which rely on regulatory elements with more tightly restricted spatial and/or temporal expression. Meiotic silencing of the sex chromosomes limits the flexibility of transgene expression to develop effective sex-linked genetic control strategies. Here, we build on our previous study, dissecting gametogenesis into four distinct cell populations, using single-cell RNA sequencing to define eight distinct cell clusters and associated germline cell-types using available marker genes. We reveal overexpression of X-linked genes in a distinct cluster of pre-meiotic cells and document the onset of meiotic silencing of the X chromosome in a subcluster of cells in the latter stages of spermatogenesis. This study provides a comprehensive dataset, characterising the expression of distinct cell types through spermatogenesis and widening the toolkit for genetic control of malaria mosquitoes.

A genetically encoded anti-CRISPR protein constrains gene drive spread and prevents population suppression.

CRISPR-based gene drives offer promising means to reduce the burden of pests and vector-borne diseases. These techniques consist of releasing genetically modified organisms carrying CRISPR-Cas nucleases designed to bias their inheritance and rapidly propagate desired modifications. Gene drives can be intended to reduce reproductive capacity of harmful insects or spread anti-pathogen effectors through wild populations, even when these confer fitness disadvantages. Technologies capable of halting the spread of gene drives may prove highly valuable in controlling, counteracting, and even reverting their effect on individual organisms as well as entire populations. Here we show engineering and testing of a genetic approach, based on the germline expression of a phage-derived anti-CRISPR protein (AcrIIA4), able to inactivate CRISPR-based gene drives and restore their inheritance to Mendelian rates in the malaria vector Anopheles gambiae. Modeling predictions and cage testing show that a single release of male mosquitoes carrying the AcrIIA4 protein can block the spread of a highly effective suppressive gene drive preventing population collapse of caged malaria mosquitoes.

The Potential for a Released Autosomal X-Shredder Becoming a Driving-Y Chromosome and Invasively Suppressing Wild Populations of Malaria Mosquitoes.

Sex-ratio distorters based on X-chromosome shredding are more efficient than sterile male releases for population suppression. X-shredding is a form of sex distortion that skews spermatogenesis of XY males towards the preferential transmission of Y-bearing gametes, resulting in a higher fraction of sons than daughters. Strains harboring X-shredders on autosomes were first developed in the malaria mosquito Anopheles gambiae, resulting in strong sex-ratio distortion. Since autosomal X-shredders are transmitted in a Mendelian fashion and can be selected against, their frequency in the population declines once releases are halted. However, unintended transfer of X-shredders to the Y-chromosome could produce an invasive meiotic drive element, that benefits from its biased transmission to the predominant male-biased offspring and its effective shielding from female negative selection. Indeed, linkage to the Y-chromosome of an active X-shredder instigated the development of the nuclease-based X-shredding system. Here, we analyze mechanisms whereby an autosomal X-shredder could become unintentionally Y-linked after release by evaluating the stability of an established X-shredder strain that is being considered for release, exploring its potential for remobilization in laboratory and wild-type genomes of An. gambiae and provide data regarding expression on the mosquito Y-chromosome. Our data suggest that an invasive X-shredder resulting from a post-release movement of such autosomal transgenes onto the Y-chromosome is unlikely.

A Code of Ethics for Gene Drive Research.

Gene drives hold promise for use in controlling insect vectors of diseases, agricultural pests, and for conservation of ecosystems against invasive species. At the same time, this technology comes with potential risks that include unknown downstream effects on entire ecosystems as well as the accidental or nefarious spread of organisms that carry the gene drive machinery. A code of ethics can be a useful tool for all parties involved in the development and regulation of gene drives and can be used to help ensure that a balanced analysis of risks, benefits, and values is taken into consideration in the interest of society and humanity. We have developed a code of ethics for gene drive research with the hope that this code will encourage the development of an international framework that includes ethical guidance of gene drive research and is incorporated into scientific practice by gaining broad agreement and adherence.

Engineered RNA-Interacting CRISPR Guide RNAs for Genetic Sensing and Diagnostics.

CRISPR guide RNAs (gRNAs) can be programmed with relative ease to allow the genetic editing of nearly any DNA or RNA sequence. Here, we propose novel molecular architectures to achieve RNA-dependent modulation of CRISPR activity in response to specific RNA molecules. We designed and tested, in both living Escherichia coli cells and cell-free assays for rapid prototyping, cis-repressed RNA-interacting guide RNA (igRNA) that switch to their active state only upon interaction with small RNA fragments or long RNA transcripts, including pathogen-derived mRNAs of medical relevance such as the human immunodeficiency virus infectivity factor. The proposed CRISPR-igRNAs are fully customizable and easily adaptable to the majority if not all the available CRISPR-Cas variants to modulate a variety of genetic functions in response to specific cellular conditions, providing orthogonal activation and increased specificity. We thereby foresee a large scope of application for therapeutic, diagnostic, and biotech applications in both prokaryotic and eukaryotic systems.

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.

Vector-Focused Approaches to Curb Malaria Transmission in the Brazilian Amazon: An Overview of Current and Future Challenges and Strategies.

In Brazil, malaria transmission is mostly confined to the Amazon, where substantial progress has been made towards disease control in the past decade. Vector control has been historically considered a fundamental part of the main malaria control programs implemented in Brazil. However, the conventional vector-control tools have been insufficient to control or eliminate local vector populations due to the complexity of the Amazonian rainforest environment and ecological features of malaria vector species in the Amazon, especially Anopheles darlingi. Malaria elimination in Brazil and worldwide eradication will require a combination of conventional and new approaches that takes into account the regional specificities of vector populations and malaria transmission dynamics. Here we present an overview on both conventional and novel promising vector-focused tools to curb malaria transmission in the Brazilian Amazon. If well designed and employed, vector-based approaches may improve the implementation of malaria-control programs, particularly in remote or difficult-to-access areas and in regions where existing interventions have been unable to eliminate disease transmission. However, much effort still has to be put into research expanding the knowledge of neotropical malaria vectors to set the steppingstones for the optimization of conventional and development of innovative vector-control tools.

Cellular mechanisms regulating synthetic sex ratio distortion in the Anopheles gambiae germline.

Genetic control strategies aimed to bias the sex of progenies towards males present a promising new paradigm to eliminate malaria-transmitting mosquitoes. A synthetic sex-ratio distortion (SD) system was successfully engineered in Anopheles gambiae by exploiting the meiotic activity of the I-PpoI endonuclease targeting ribosomal DNA (rDNA) repeats, exclusively located on the X chromosome. Males carrying the SD construct produce highly male-biased progenies without evident reduction in fertility. In this study, we investigated the fate of X and Y chromosomes in these SD males and found that ratios of mature X:Y-bearing sperm were comparable to wild-type insects, indicating absence of selection mechanisms during sperm maturation. We therefore tested the effect of meiotic cleavage of both X and Y chromosomes in a lab-generated SD strain carrying rDNA on both sex chromosomes, showing fertility comparable to wild-type and a reduced male-bias compared to SD males in which only the X is targeted. Exposure of Y-linked rDNA to I-PpoI cleavage for consecutive generations rapidly restored the male-bias to typical high frequencies, indicating a correlation between the number of cleavable targets in each sex chromosome and the sex-ratios found in the progeny. Altogether our results indicate that meiotic cleavage of rDNA repeats, located in the sex chromosomes of A. gambiae SD males, affects the competitiveness of mature sperm to fertilize the female oocyte, thereby generating sex-biased progenies. We also show that the presence of rDNA copies on the Y chromosome does not impair the effectiveness of engineered synthetic SD systems for the control of human malaria mosquitoes.