Biology subjects

Crisanti, A.

Publications and source records attributed to Crisanti, A..

13 recordsLinked to original sources

bioRxiv2026

Essential function of femaleless in female gametogenesis controls gene drive spread in Anopheles gambiae

Insecticide resistance in mosquito vectors and antimalarial drug resistance in parasites threaten progress towards malaria elimination, prompting the development of alternative control strategies such as CRISPR-Cas9 gene drives. The sex determination gene femaleless (fle, AGAP013051), which is required for female development in Anopheles gambiae, is a promising target for population-suppression approaches aimed at disrupting female-specific genes that affect fertility or viability. However, its functions beyond sex determination remain unknown. Here, we engineered homing gene drives targeting fle and employed germline promoters with distinct temporal expression profiles, early-acting , zero population growth (zpg, AGAP006241) and late-acting sporulation defective 11 (spo11, AGAP010898), to modulate Cas9 activity. The zpg-driven system achieved up to 98% transmission through males but caused complete sterility in hemizygous females due to early biallelic disruption of fle during germline development. Delaying cas9 expression with the spo11 promoter partially restored female fertility, although female transmission remained close to Mendelian levels (59%). These results reveal an essential role for fle in female gametogenesis in addition to its established function in sex determination. Population modelling predicts that releasing zpg-drive males at 16.9% of the wild-type male population could reduce female abundance by 95% within 36 generations. Collectively, our findings reveal a previously unrecognised reproductive function of fle that limits gene-drive spread and provide important insights for the design of vector-control strategies targeting genes with essential germline functions.

synthetic biology↗

bioRxiv2025

A male-drive female-sterile system for the self-limited control of the malaria mosquito Anopheles gambiae

The use of insecticides and antimalarial drugs has been crucial in reducing the mortality and morbidity associated with malaria. However, since 2015, several challenges, including the development of resistance to these insecticides and treatments and changes in mosquito behaviour, have hindered the progress in fighting the disease. As a result, there is a pressing need for new tools to control malaria, including the potential use of genetically modified mosquitoes (GMMs) in the field. Various genetic strategies for vector control are currently being explored, ranging from self-sustaining GMMs with unrestricted geographic and temporal spread to self-limiting alternatives. Here, we propose a self-limiting gene drive strategy called Male Drive Female Sterile (MDFS) targeting Anopheles gambiae, a major malaria vector. The MDFS genetic construct causes dominant sterility in females, while transgenic males remain fertile, allowing them to transmit the female sterility trait at super-Mendelian rates. Laboratory studies have shown that repeated releases of MDFS can lead to the elimination of caged mosquito populations. Based on these findings, modelling suggests that MDFS could be a highly effective and self-limiting strategy for suppressing wild malaria mosquito populations.

molecular biology↗

bioRxiv2025

Using a female-specific isoform of doublesex to explore male-specific hearing in mosquitoes

Animal reproduction relies on elaborate divisions of labour and multiple dimorphisms between the sexes. Primary dimorphisms affect core elements of reproduction, secondary dimorphisms affect more indirect traits, including complex behaviours. In disease-transmitting mosquitoes, males locate females acoustically prior to copulation (phonotaxis). No comparable acoustic behaviour is known for females. As a result, the males ears - and hearing performance - have evolved to become substantially more complex. Sex-specific hearing in mosquitoes is in part controlled by the doublesex (dsx) gene. Intriguingly, dsx forms a linker between primary and secondary dimorphisms: spermatogenesis and ear morphogenesis share considerable molecular overlap and both depend on dsx expression patterns. We have combined transcriptomics with functional-anatomical analyses to dissect dsx-dependent hearing in the malaria mosquito Anopheles gambiae. By cross-linking our auditory findings to the genetic bases of spermatogenesis we advance the molecular understanding of sex-specific hearing mechanisms in insects, highlighting the special roles of ciliary factors therein. HighlightsO_LIDisruption of the female-specific exon of the doublesex gene alters hearing in female Anopheles gambiae mosquitoes C_LIO_LIFemale mutant ears are anatomically and functionally distinct from both wild-type male and female; they show a substantial, but incomplete, masculinisation C_LIO_LIContrary to males, and similarly to females, the flagella of female mutants do not exhibit Self-Sustained Oscillations (SSOs) C_LIO_LITranscriptomic comparisons of ears uncovered molecular bases of auditory maleness, these were closely linked to molecular machinery of ciliary function C_LIO_LIComparisons with existing transcriptome from male testes revealed a substantial overlap between male ear and testicular ciliary motility machineries C_LI

neuroscience↗

bioRxiv2024

Engineering Resilient Gene Drives Towards Sustainable Malaria Control: Predicting, Testing and Overcoming Target Site Resistance

CRISPR-based gene drives are selfish genetic elements with the potential to spread through entire insect populations for sustainable vector control. Gene drives designed to disrupt the reproductive capacity of females can suppress laboratory populations of the malaria mosquito. However, any suppressive intervention will inevitably exert an evolutionary pressure for resistance. Here, we present a pipeline for the accelerated discovery, engineering, and testing of both natural and drive-induced variants that could reverse gene drive spread. We applied our method to stress-test a highly effective gene drive that has evaded resistance in all laboratory-contained releases to date, known as Ag(QFS)1. We showed that previously undetected resistant alleles can arise at low frequency, and discovered novel, partially resistant alleles that can perturb drive-invasion dynamics. We then engineered next-generation gene drives that can actively remove resistant alleles by targeting several highly conserved and non-overlapping sites in the female-specific exon of the doublesex gene. Our models predict that such gene drive designs could suppress large, natural populations of the malaria mosquito in the field.

synthetic biology↗

bioRxiv2024

A Y chromosome-linked genome editor for efficient population suppression in the malaria vector Anopheles gambiae

Genetic control - the deliberate introduction of genetic traits to control a pest or vector population - offers a powerful tool to augment conventional mosquito control tools that have been successful in reducing malaria burden but that are compromised by a range of operational challenges. Self-sustaining genetic control strategies have shown great potential in laboratory settings but hesitancy due to their invasive and persistent nature may delay their implementation. Here instead we describe a self-limiting strategy, designed to have geographically and/or temporally restricted effect, based on a Y chromosome-linked genome editor (YLE). The YLE comprises a CRISPR-Cas9 construct that is always inherited by males yet generates an autosomal dominant mutation that is transmitted to over 90% of the offspring and results in female-specific sterility. Males are unaffected. To our knowledge, our system represents the first engineering of the Y chromosome to generate a genetic control strain for mosquitoes. Mathematical modelling shows that this YLE technology is up to 8 times more efficient for population suppression than optimal versions of other self-limiting strategies.

synthetic biology↗

bioRxiv2021

Rapid longitudinal SARS-CoV-2 intra-host emergence of novel haplotypes regardless of immune deficiencies

On February 2020, the municipality of Vo, a small town near Padua (Italy), was quarantined due to the first coronavirus disease 19 (COVID-19)-related death detected in Italy. The entire population was swab tested in two sequential surveys. Here we report the analysis of the viral genomes, which revealed that the unique ancestor haplotype introduced in Vo belongs to lineage B and, more specifically, to the subtype found at the end of January 2020 in two Chinese tourists visiting Rome and other Italian cities, carrying mutations G11083T and G26144T. The sequences, obtained for 87 samples, allowed us to investigate viral evolution while being transmitted within and across households and the effectiveness of the non-pharmaceutical interventions implemented in Vo. We report, for the first time, evidence that novel viral haplotypes can naturally arise intra-host within an interval as short as two weeks, in approximately 30% of the infected individuals, regardless of symptoms severity or immune system deficiencies. Moreover, both phylogenetic and minimum spanning network analyses converge on the hypothesis that the viral sequences evolved from a unique common ancestor haplotype, carried by an index case. The lockdown extinguished both viral spread and the emergence of new variants, confirming the efficiency of this containment strategy. The information gathered from household was used to reconstructs possible transmission events. AUTHOR SUMMARYIt is of great interest and importance to understand SARS-CoV-2 ability to mutate generating new viral strains, and to assess the impact of containment strategies on viral transmission. In this study we highlight the rapid intra-host haplotype evolution regardless of symptom severity and immune deficiencies that we observed during the first wave of the pandemic in the municipality of Vo in Italy. The confirmation that all the haplotypes found in this small community derive from a common ancestor haplotype, has allowed us to track the rapid emergence of new variants but lockdown and mass testing efficiently prevented their spread elsewhere.

evolutionary biology↗

bioRxiv2021

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 gene drive 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. Author summaryGene drives have the potential to be applied as novel control strategy of disease-transmitting mosquitoes, by spreading genetic traits that suppress or modify the target population. Many gene drive elements work by recognising and cutting a specific target sequence in the mosquito genome and copying themselves into that target sequence allowing the gene drive to increase in frequency in the population. Like other mosquito control interventions, efficacy will greatly depend on minimising the development of resistance to the gene drive mechanism - most likely via a change in the target sequence that prevents further cutting. One strategy to reduce resistance is to target sequences that are highly conserved, which implies that changes cannot easily be tolerated. We developed a strategy that simulates high selection pressure, under which resistance is most likely to emerge, and therefore provides a stringent test of its propensity to arise. Unlike previous results with another gene drive, we recovered a resistant allele within a few generations of gene drive exposure and at high frequency. Our results show that conserved sequences can vary hugely in ability to tolerate mutations and highlights the need to functionally validate future candidate gene drive target sites for their robustness to resistance.

genetics↗

bioRxiv2021

Ultra-conserved sequences in the genomes of highly diverse Anopheles mosquitoes, with implications for malaria vector control.

DNA sequences that are exactly conserved over long evolutionary time scales have been observed in a variety of taxa. Such sequences are likely under strong functional constraint and they have been useful in the field of comparative genomics for identifying genome regions with regulatory function. A potential new application for these ultra-conserved elements has emerged in the development of gene drives to control mosquito populations. Many gene drives work by recognising and inserting at a specific target sequence in the genome, often imposing a reproductive load as a consequence. They can therefore select for target sequence variants that provide resistance to the drive. Focusing on highly conserved, highly constrained sequences lowers the probability that variant, gene drive-resistant alleles can be tolerated. Here we search for conserved sequences of 18bp and over in an alignment of 21 Anopheles genomes, spanning an evolutionary timescale of 100 million years, and characterise the resulting sequences according to their location and function. Over 8000 ultra-conserved elements were found across the alignment, with a maximum length of 164 bp. Length-corrected gene ontology analysis revealed that genes containing Anopheles ultra-conserved elements were over-represented in categories with structural or nucleotide binding functions. Known insect transcription factor binding sites were found in 48% of intergenic Anopheles ultra-conserved elements. When we looked at the genome sequences of 1142 wild-caught mosquitoes we found that 15% of the Anopheles ultra-conserved elements contained no polymorphisms. Our list of Anopheles ultra-conserved elements should provide a valuable starting point for the selection and testing of new targets for gene-drive modification in the mosquitoes that transmit malaria.

genomics↗

bioRxiv2020

Assessing the acoustic behaviour of Anopheles gambiae s.l. dsxF mutants: Implications for Vector Control

BackgroundThe release of genetically modified mosquitoes which use gene-drive mechanisms to suppress reproduction in natural populations of Anopheles mosquitoes is one of the scientifically most promising methods for malaria transmission control. However, many scientific, regulatory and ethical questions remain before transgenic mosquitoes can be utilised in the field. Mutations which reduce an individuals reproductive success are likely to create strong selective pressures to evolve resistance. It is thus crucial that the targeted population collapses as rapidly and as completely as possible to reduce the available time for the emergence of drive-resistant mutations. At a behavioural level, this means that the gene-drive carrying mutants should be at least as (and ideally more) sexually attractive than the wildtype population they compete against. A key element in the copulatory negotiations of Anopheles mosquitoes is their acoustic courtship. We therefore analysed sound emissions and acoustic preference in a doublesex mutant previously used to successfully collapse caged colonies of Anopheles gambiae s.l.. MethodsThe flight tones produced by the beating of their wings form the signals for acoustic mating communication in Anopheles species. We assessed the acoustic impact of the disruption of a female-specific isoform of the doublesex gene (dsxF) on the wing beat frequency (WBF; measured as flight tone) of both males (XY) and females (XX) in homozygous dsxF- mutants (dsxF-/-), heterozygous dsxF- carriers (dsxF+/-) and G3 wildtype dsxF+ controls (dsxF+/+). To exclude non-genetic influences, we controlled for temperature and measured wing lengths for all experimental animals. We used a phonotaxis assay to test the acoustic preferences of mutant and control mosquitoes. ResultsA previous study demonstrated an altered phenotype only for females homozygous for the disrupted dsx allele (dsxF-/-), who appear intersex. No phenotypic changes were observed for heterozygous carriers or males, suggesting that the female-specific dsxF allele is haplosufficient. We here identify significant, dose-dependent increases in the flight tones of both dsxF-/- and dsxF+/- females when compared to dsxF+/+ control females. Flight tone frequencies in all three female genotypes remained significantly lower than in males, however. When tested experimentally, males showed stronger phonotactic responses to the flight tones of control dsxF+/+ females. While flight tones from dsxF+/- and dsxF-/- females also elicited positive phonotactic behaviour in males, this was significantly reduced compared to responses to control tones. We found no evidence of phonotactic behaviour in any female genotype tested. None of the male genotypes displayed any deviations from the control condition. ConclusionsA key prerequisite for copulation in anopheline mosquitoes is the phonotactic attraction of males towards female flight tones within large - spatially and acoustically crowded - mating swarms. Reductions in acoustic attractiveness of released mutant lines, as reported here for heterozygous dsxF+/- females, reduce the lines mating efficiency, and could consequently reduce the efficacy of the associated population control effort. Assessments of caged populations may not successfully reproduce the challenges posed by natural mating scenarios. We propose to amend existing testing protocols in order to more faithfully reflect the competitive conditions between a mutant line and the wildtype population it is meant to interact with. This should also include novel tests of acoustic fitness. In line with previous studies, our findings confirm that disruption of the female-specific isoform dsxF has no effect on males; for some phenotypic traits, such as female flight tones, however, the effects of dsxF appear to be dose-dependent rather than haplosufficient.

animal behavior and cognition↗

bioRxiv2020

A litmus test for harmonized mosquito monitoring across Europe and Africa

The accelerating rate of outbreaks from mosquito borne diseases are urging the development of updated and effective tools for the surveillance of insect populations and their larval habitats. Harmonized field protocols help to build a comprehensive picture on species-specific vector ecology and generate important knowledge for implementing coordinated mosquito surveillance programs at regional scales and across continents. In this study, we test the efficiency and potential barriers of available harmonized protocols from earlier EU project VectorNet. As a kind of litmus test for such protocols, we specifically aim to capture the ecoregional variation of breeding site characteristics and population density of five mosquito vectors in Europe and Africa. As expected, the five species considered show different aquatic habitat preferences. Culex pipiens, Aedes albopictus in Europe and Ae. aegypti in Africa select breeding habitats within specific volume classes, while Anopheles gambiae and An. coluzzii may select breeding habitats based on seasonal availability. Population densities in aquatic habitats greatly varied across species and countries, but larval production sites of Ae. albopictus generate populations with higher ratio of pupae compared to the other species. This result underlines the fundamental ecological difference between the selected vector species disregarding of the ecoregion. Mean water temperatures had limited variation across species and higher among countries. Understanding the ecology of native and non-native mosquito vectors is key in evaluating transmission risks for diseases such as West Nile, chikungunya and dengue fevers, zika and malaria. The available harmonized field protocols are a valuable tool for achieving homogeneous mosquito surveillance in Europe and Africa.

ecology↗

bioRxiv2018

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

CRISPR-Cas9 nuclease-based gene drives rely on inducing chromosomal breaks in the germline that are repaired in ways that lead to a biased inheritance of the drive. Gene drives designed to impair female fertility can suppress populations of the mosquito vector of malaria. However, strong unintended fitness costs, due to ectopic nuclease expression, and high levels of resistant mutations, limited the potential of the first generation of gene drives to spread. Here we show that changes to regulatory sequences in the drive element, designed to contain nuclease expression to the germline, confer improved fecundity over previous versions and generate drastically lower rates of target site resistance. We employed a genetic screen to show that this effect is explained by reduced rates of end-joining repair of DNA breaks at the target site caused by deposited nuclease in the embryo. Highlighting the impact of deposited Cas9, many of the mutations arising from this source of nuclease activity in the embryo are heritable, thereby having the potential to generate resistant target sites that reduce the penetrance of the gene drive. Finally, in cage invasion experiments these gene drives show improved invasion dynamics compared to 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 resistance-prone target sequence. We shed light on the dynamics of generation and selection of resistant alleles in a population by tracking, longitudinally, the frequency of resistant alleles in the face of an invading gene drive. Our results illustrate important considerations for future gene drive design and should expedite the development of gene drives robust to resistance.

genetics↗

bioRxiv2017

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 F1xF0 crossing scheme to overcome a severe bottleneck of male hybrid incompatibilities and enabled us to experimentally purify a genetically labelled A. gambiae Y chromosome in an A. arabiensis background. Whole genome sequencing 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 1.85Myr ago 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.

genetics↗

bioRxiv2017

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 on the population.\n\nWe have recently developed gene drives based on a CRISPR nuclease construct that is 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, in 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 noticed a gradual decrease in its frequency that was accompanied emergence of small, nuclease-induced mutations at the target gene that are resistant to further cleavage and restore its functionality. Such mutations show 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 for future gene drive applications.

evolutionary biology↗