Roberto Galizi

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.

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.

Temporal and Spatial Distribution of Toxoplasma gondii Differentiation into Bradyzoites and Tissue Cyst Formation In Vivo

ABSTRACT During Toxoplasma gondii infection, a fraction of the multiplying parasites, the tachyzoites, converts into bradyzoites, a dormant stage, which form tissue cysts localized mainly in brain, heart, and skeletal muscles that persist for several years after infection. At this stage the parasite is protected from the immune system, and it is believed to be inaccessible to drugs. While the long persistence of tissue cysts does not represent a medical problem for healthy individuals, this condition represents a major risk for patients with a compromised immune system, who can develop recrudescent life-threatening T. gondii infections. We have investigated for the first time the dynamics and the kinetics of tachyzoite-to-bradyzoite interconversion and cyst formation in vivo by using stage-specific bioluminescent parasites in a mouse model. Our findings provide a new framework for understanding the process of bradyzoite differentiation in vivo. We have also demonstrated that complex molecules such as d-luciferin have access to tissue cysts and are metabolically processed, thus providing a rationale for developing drugs that attack the parasite at this developmental stage.

Site-specific genetic engineering of the Anopheles gambiae Y chromosome

Significance Interfering with sex determination and male fertility are potentially powerful approaches for the genetic control of the human malaria vector Anopheles gambiae. Despite this fact, the male-specific Y chromosome of this mosquito has remained largely unexplored, because of its repetitive, heterochromatic structure. Little is known about its ability to support gene transcription in different tissues and during gametogenesis, yet this information is crucial for understanding the function of this chromosome. We show, using a combination of knock-in and site-specific genetic engineering steps, how transgenes can be specifically introduced onto the Y chromosome. The Y-linked strains we have created provide the means to generate large single-sex populations and to establish male-exclusive genetic traits for the control of this important vector species. 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.

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

Significance Interest in male mosquitoes has been motivated by the potential to develop novel vector control strategies, exploiting the fact that males do not feed on blood or transmit diseases, such as malaria. However, genetic studies of male Anopheles mosquitoes have been impeded by the lack of molecular characterization of the Y chromosome. Here we show that the Anopheles gambiae Y chromosome contains a very small repertoire of genes, with massively amplified tandem arrays of a small number of satellites and transposable elements constituting the vast majority of the sequence. These genes and repeats evolve rapidly, bringing about remodeling of the Y, even among closely related species. Our study provides a long-awaited foundation for studying mosquito Y chromosome biology and evolution. 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.

A CRISPR-Cas9 sex-ratio distortion system for genetic control.

Genetic control aims to reduce the ability of insect pest populations to cause harm via the release of modified insects. One strategy is to bias the reproductive sex ratio towards males so that a population decreases in size or is eliminated altogether due to a lack of females. We have shown previously that sex ratio distortion can be generated synthetically in the main human malaria vector Anopheles gambiae, by selectively destroying the X-chromosome during spermatogenesis, through the activity of a naturally-occurring endonuclease that targets a repetitive rDNA sequence highly-conserved in a wide range of organisms. Here we describe a CRISPR-Cas9 sex distortion system that targets ribosomal sequences restricted to the member species of the Anopheles gambiae complex. Expression of Cas9 during spermatogenesis resulted in RNA-guided shredding of the X-chromosome during male meiosis and produced extreme male bias among progeny in the absence of any significant reduction in fertility. The flexibility of CRISPR-Cas9 combined with the availability of genomic data for a range of insects renders this strategy broadly applicable for the species-specific control of any pest or vector species with an XY sex-determination system by targeting sequences exclusive to the female sex chromosome.

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.

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

BackgroundSmall 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.ResultsWe 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.ConclusionsThis 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.

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.

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.