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1.
Mol Syst Biol ; 2024 Jul 28.
Artigo em Inglês | MEDLINE | ID: mdl-39069594

RESUMO

Secretion systems play a crucial role in microbe-microbe or host-microbe interactions. Among these systems, the extracellular contractile injection system (eCIS) is a unique bacterial and archaeal extracellular secretion system that injects protein toxins into target organisms. However, the specific proteins that eCISs inject into target cells and their functions remain largely unknown. Here, we developed a machine learning classifier to identify eCIS-associated toxins (EATs). The classifier combines genetic and biochemical features to identify EATs. We also developed a score for the eCIS N-terminal signal peptide to predict EAT loading. Using the classifier we classified 2,194 genes from 950 genomes as putative EATs. We validated four new EATs, EAT14-17, showing toxicity in bacterial and eukaryotic cells, and identified residues of their respective active sites that are critical for toxicity. Finally, we show that EAT14 inhibits mitogenic signaling in human cells. Our study provides insights into the diversity and functions of EATs and demonstrates machine learning capability of identifying novel toxins. The toxins can be employed in various applications dependently or independently of eCIS.

2.
Nat Commun ; 15(1): 4983, 2024 Jun 11.
Artigo em Inglês | MEDLINE | ID: mdl-38862555

RESUMO

Engineered sex ratio distorters (SRDs) have been proposed as a powerful component of genetic control strategies designed to suppress harmful insect pests. Two types of CRISPR-based SRD mechanisms have been proposed: X-shredding, which eliminates X-bearing sperm, and X-poisoning, which eliminates females inheriting disrupted X-chromosomes. These differences can have a profound impact on the population dynamics of SRDs when linked to the Y-chromosome: an X-shredder is invasive, constituting a classical meiotic Y-drive, whereas X-poisoning is self-limiting, unable to invade but also insulated from selection. Here, we establish X-poisoning strains in the malaria vector Anopheles gambiae targeting three X-linked genes during spermatogenesis, resulting in male bias. We find that sex distortion is primarily driven by a loss of X-bearing sperm, with limited evidence for postzygotic lethality of female progeny. By leveraging a Drosophila melanogaster model, we show unambiguously that engineered SRD traits can operate differently in these two insects. Unlike X-shredding, X-poisoning could theoretically operate at early stages of spermatogenesis. We therefore explore premeiotic Cas9 expression to target the mosquito X-chromosome. We find that, by pre-empting the onset of meiotic sex chromosome inactivation, this approach may enable the development of Y-linked SRDs if mutagenesis of spermatogenesis-essential genes is functionally balanced.


Assuntos
Anopheles , Drosophila melanogaster , Tecnologia de Impulso Genético , Razão de Masculinidade , Espermatogênese , Cromossomo X , Animais , Masculino , Feminino , Anopheles/genética , Cromossomo X/genética , Drosophila melanogaster/genética , Tecnologia de Impulso Genético/métodos , Espermatogênese/genética , Mosquitos Vetores/genética , Genes Ligados ao Cromossomo X , Sistemas CRISPR-Cas , Espermatozoides/metabolismo , Animais Geneticamente Modificados
3.
Nat Commun ; 15(1): 372, 2024 Jan 08.
Artigo em Inglês | MEDLINE | ID: mdl-38191463

RESUMO

Homing-based gene drives are recently proposed interventions promising the area-wide, species-specific genetic control of harmful insect populations. Here we characterise a first set of gene drives in a tephritid agricultural pest species, the Mediterranean fruit fly Ceratitis capitata (medfly). Our results show that the medfly is highly amenable to homing-based gene drive strategies. By targeting the medfly transformer gene, we also demonstrate how CRISPR-Cas9 gene drive can be coupled to sex conversion, whereby genetic females are transformed into fertile and harmless XX males. Given this unique malleability of sex determination, we modelled gene drive interventions that couple sex conversion and female sterility and found that such approaches could be effective and tolerant of resistant allele selection in the target population. Our results open the door for developing gene drive strains for the population suppression of the medfly and related tephritid pests by co-targeting female reproduction and shifting the reproductive sex ratio towards males. They demonstrate the untapped potential for gene drives to tackle agricultural pests in an environmentally friendly and economical way.


Assuntos
Ceratitis capitata , Tecnologia de Impulso Genético , Feminino , Masculino , Animais , Ceratitis capitata/genética , Agricultura , Alelos , Fontes de Energia Elétrica
4.
bioRxiv ; 2023 Aug 04.
Artigo em Inglês | MEDLINE | ID: mdl-37577635

RESUMO

Insects have developed remarkable adaptations to effectively interact with plant secondary metabolites and utilize them as cues to identify suitable hosts. Consequently, humans have used aromatic plants for centuries to repel mosquitoes. The repellent effects of plant volatile compounds are mediated through olfactory structures present in the antennae, and maxillary palps of mosquitoes. Mosquito maxillary palps contain capitate-peg sensilla, which house three olfactory sensory neurons, of which two are mainly tuned to either carbon dioxide or octenol - two animal host odorants. However, the third neuron, which expresses the OR49 receptor, has remained without a known ecologically-relevant odorant since its initial discovery. In this study, we used odorant mixtures and terpenoid-rich Cannabis essential oils to investigate the activation of OR49. Our results demonstrate that two monoterpenoids, borneol and camphor, selectively activate OR49, and OR9-expressing neurons, as well as the MD3 glomerulus in the antennal lobe. We confirm that borneol repels female mosquitoes, and knocking out the gene encoding the OR49 receptor suppresses the response of the corresponding olfactory sensory neuron. Importantly, this molecular mechanism of action is conserved across culicine mosquito species, underscoring its significance in their olfactory systems.

5.
DNA Res ; 30(1)2023 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-36370138

RESUMO

The New World Screwworm, Cochliomyia hominivorax (Calliphoridae), is the most important myiasis-causing species in America. Screwworm myiasis is a zoonosis that can cause severe lesions in livestock, domesticated and wild animals, and occasionally in people. Beyond the sanitary problems associated with this species, these infestations negatively impact economic sectors, such as the cattle industry. Here, we present a chromosome-scale assembly of C. hominivorax's genome, organized in 6 chromosome-length and 515 unplaced scaffolds spanning 534 Mb. There was a clear correspondence between the D. melanogaster linkage groups A-E and the chromosomal-scale scaffolds. Chromosome quotient (CQ) analysis identified a single scaffold from the X chromosome that contains most of the orthologs of genes that are on the D. melanogaster fourth chromosome (linkage group F or dot chromosome). CQ analysis also identified potential X and Y unplaced scaffolds and genes. Y-linkage for selected regions was confirmed by PCR with male and female DNA. Some of the long chromosome-scale scaffolds include Y-linked sequences, suggesting misassembly of these regions. These resources will provide a basis for future studies aiming at understanding the biology and evolution of this devastating obligate parasite.


Assuntos
Miíase , Infecção por Mosca da Bicheira , Animais , Masculino , Feminino , Bovinos , Calliphoridae , Drosophila melanogaster , Miíase/veterinária , Infecção por Mosca da Bicheira/veterinária , Cromossomos
6.
Front Bioeng Biotechnol ; 9: 752253, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34957064

RESUMO

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.

7.
Nat Commun ; 12(1): 7202, 2021 12 10.
Artigo em Inglês | MEDLINE | ID: mdl-34893590

RESUMO

CRISPR-based genetic engineering tools aimed to bias sex ratios, or drive effector genes into animal populations, often integrate the transgenes into autosomal chromosomes. However, in species with heterogametic sex chromsomes (e.g. XY, ZW), sex linkage of endonucleases could be beneficial to drive the expression in a sex-specific manner to produce genetic sexing systems, sex ratio distorters, or even sex-specific gene drives, for example. To explore this possibility, here we develop a transgenic line of Drosophila melanogaster expressing Cas9 from the Y chromosome. We functionally characterize the utility of this strain for both sex selection and gene drive finding it to be quite effective. To explore its utility for population control, we built mathematical models illustrating its dynamics as compared to other state-of-the-art systems designed for both population modification and suppression. Taken together, our results contribute to the development of current CRISPR genetic control tools and demonstrate the utility of using sex-linked Cas9 strains for genetic control of animals.


Assuntos
Sistemas CRISPR-Cas , Tecnologia de Impulso Genético/métodos , Genes Ligados ao Cromossomo Y , Pré-Seleção do Sexo/métodos , Cromossomo Y , Animais , Animais Geneticamente Modificados , Drosophila melanogaster/genética , Endonucleases/genética , Feminino , Edição de Genes/métodos , Masculino , Razão de Masculinidade , Biologia Sintética/métodos , Transgenes
9.
BMC Biol ; 19(1): 78, 2021 04 16.
Artigo em Inglês | MEDLINE | ID: mdl-33863334

RESUMO

BACKGROUND: Genetic sex ratio distorters are systems aimed at effecting a bias in the reproductive sex ratio of a population and could be applied for the area-wide control of sexually reproducing insects that vector disease or disrupt agricultural production. One example of such a system leading to male bias is X-shredding, an approach that interferes with the transmission of the X-chromosome by inducing multiple DNA double-strand breaks during male meiosis. Endonucleases targeting the X-chromosome and whose activity is restricted to male gametogenesis have recently been pioneered as a means to engineer such traits. RESULTS: Here, we enabled endogenous CRISPR/Cas9 and CRISPR/Cas12a activity during spermatogenesis of the Mediterranean fruit fly Ceratitis capitata, a worldwide agricultural pest of extensive economic significance. In the absence of a chromosome-level assembly, we analysed long- and short-read genome sequencing data from males and females to identify two clusters of abundant and X-chromosome-specific sequence repeats. When targeted by gRNAs in conjunction with Cas9, cleavage of these repeats yielded a significant and consistent distortion of the sex ratio towards males in independent transgenic strains, while the combination of distinct distorters induced a strong bias (~ 80%). CONCLUSION: We provide a first demonstration of CRISPR-based sex distortion towards male bias in a non-model organism, the global pest insect Ceratitis capitata. Although the sex ratio bias reached in our study would require improvement, possibly through the generation and combination of additional transgenic lines, to result in a system with realistic applicability in the field, our results suggest that strains with characteristics suitable for field application can now be developed for a range of medically or agriculturally relevant insect species.


Assuntos
Ceratitis capitata , Animais , Animais Geneticamente Modificados , Sistemas CRISPR-Cas/genética , Ceratitis capitata/genética , Feminino , Masculino , RNA Guia de Cinetoplastídeos , Razão de Masculinidade , Cromossomo X/genética
11.
J Appl Ecol ; 57(10): 2086-2096, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-33149368

RESUMO

The development of genetically modified (GM) mosquitoes and their subsequent field release offers innovative and cost-effective approaches to reduce mosquito-borne diseases, such as malaria. A sex-distorting autosomal transgene has been developed recently in G3 mosquitoes, a laboratory strain of the malaria vector Anopheles gambiae s.l. The transgene expresses an endonuclease called I-PpoI during spermatogenesis, which selectively cleaves the X chromosome to result in ~95% male progeny. Following the World Health Organization guidance framework for the testing of GM mosquitoes, we assessed the dynamics of this transgene in large cages using a joint experimental modelling approach.We performed a 4-month experiment in large, indoor cages to study the population genetics of the transgene. The cages were set up to mimic a simple tropical environment with a diurnal light-cycle, constant temperature and constant humidity. We allowed the generations to overlap to engender a stable age structure in the populations. We constructed a model to mimic the experiments, and used the experimental data to infer the key model parameters.We identified two fitness costs associated with the transgene. First, transgenic adult males have reduced fertility and, second, their female progeny have reduced pupal survival rates. Our results demonstrate that the transgene is likely to disappear in <3 years under our confined conditions. Model predictions suggest this will be true over a wide range of background population sizes and transgene introduction rates. Synthesis and applications. Our study is in line with the World Health Organization guidance recommendations in regard to the development and testing of GM mosquitoes. Since the transgenic sex ratio distorter strain (Ag(PMB)1) has been considered for genetic vector control of malaria, we recorded the dynamics of this transgene in indoor-large cage populations and modelled its post-release persistence under different scenarios. We provide a demonstration of the self-limiting nature of the transgene, and identified new fitness costs that will further reduce the longevity of the transgene after its release. Finally, our study has showcased an alternative and effective statistical method for characterizing the phenotypic expression of a transgene in an insect pest population.

12.
Genome Biol ; 21(1): 215, 2020 08 26.
Artigo em Inglês | MEDLINE | ID: mdl-32847630

RESUMO

BACKGROUND: The Asian tiger mosquito Aedes albopictus is globally expanding and has become the main vector for human arboviruses in Europe. With limited antiviral drugs and vaccines available, vector control is the primary approach to prevent mosquito-borne diseases. A reliable and accurate DNA sequence of the Ae. albopictus genome is essential to develop new approaches that involve genetic manipulation of mosquitoes. RESULTS: We use long-read sequencing methods and modern scaffolding techniques (PacBio, 10X, and Hi-C) to produce AalbF2, a dramatically improved assembly of the Ae. albopictus genome. AalbF2 reveals widespread viral insertions, novel microRNAs and piRNA clusters, the sex-determining locus, and new immunity genes, and enables genome-wide studies of geographically diverse Ae. albopictus populations and analyses of the developmental and stage-dependent network of expression data. Additionally, we build the first physical map for this species with 75% of the assembled genome anchored to the chromosomes. CONCLUSION: The AalbF2 genome assembly represents the most up-to-date collective knowledge of the Ae. albopictus genome. These resources represent a foundation to improve understanding of the adaptation potential and the epidemiological relevance of this species and foster the development of innovative control measures.


Assuntos
Aedes/genética , Arbovírus/genética , Genoma , Mosquitos Vetores/genética , Aedes/imunologia , Aedes/virologia , Animais , Mapeamento Cromossômico , Cromossomos , Tamanho do Genoma , Imunidade , Insetos Vetores , Mosquitos Vetores/imunologia , Mosquitos Vetores/virologia , RNA Interferente Pequeno/genética , Transcriptoma
13.
PLoS Genet ; 16(3): e1008647, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32168334

RESUMO

Synthetic sex distorters have recently been developed in the malaria mosquito, relying on endonucleases that target the X-chromosome during spermatogenesis. Although inspired by naturally-occurring traits, it has remained unclear how they function and, given their potential for genetic control, how portable this strategy is across species. We established Drosophila models for two distinct mechanisms for CRISPR/Cas9 sex-ratio distortion-"X-shredding" and "X-poisoning"-and dissected their target-site requirements and repair dynamics. X-shredding resulted in sex distortion when Cas9 endonuclease activity occurred during the meiotic stages of spermatogenesis but not when Cas9 was expressed from the stem cell stages onwards. Our results suggest that X-shredding is counteracted by the NHEJ DNA repair pathway and can operate on a single repeat cluster of non-essential sequences, although the targeting of a number of such repeats had no effect on the sex ratio. X-poisoning by contrast, i.e. targeting putative haplolethal genes on the X chromosome, induced a high bias towards males (>92%) when we directed Cas9 cleavage to the X-linked ribosomal target gene RpS6. In the case of X-poisoning sex distortion was coupled to a loss in reproductive output, although a dominant-negative effect appeared to drive the mechanism of female lethality. These model systems will guide the study and the application of sex distorters to medically or agriculturally important insect target species.


Assuntos
Edição de Genes/métodos , Processos de Determinação Sexual/genética , Pré-Seleção do Sexo/métodos , Animais , Sistemas CRISPR-Cas/genética , Reparo do DNA por Junção de Extremidades/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Endonucleases/genética , Feminino , Masculino , Modelos Animais , Controle Biológico de Vetores/métodos , Razão de Masculinidade , Espermatogênese/genética , Cromossomo X/genética
14.
Arch Insect Biochem Physiol ; 103(3): e21652, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31845410

RESUMO

Agricultural pest control using genetic-based methods provides a species-specific and environmentally harmless way for population suppression of fruit flies. One way to improve the efficiency of such methods is through self-limiting, female-eliminating approaches that can alter an insect populations' sex ratio toward males. In this microreview, we summarize recent advances in synthetic sex ratio distorters based on X-chromosome shredding that can induce male-biased progeny. We outline the basic principles to guide the efficient design of an X-shredding system in an XY heterogametic fruit fly species of interest using CRISPR/Cas gene editing, newly developed computational tools, and insect genetic engineering. We also discuss technical aspects and challenges associated with the efficient transferability of this technology in fruit fly pest populations, toward the potential use of this new class of genetic control approaches for pest management purposes.


Assuntos
Sistemas CRISPR-Cas , Dípteros/genética , Controle de Insetos/métodos , Controle Biológico de Vetores/métodos , Razão de Masculinidade , Animais , Feminino , Edição de Genes , Masculino
15.
Science ; 365(6460): 1457-1460, 2019 09 27.
Artigo em Inglês | MEDLINE | ID: mdl-31467189

RESUMO

In insects, rapidly evolving primary sex-determining signals are transduced by a conserved regulatory module controlling sexual differentiation. In the agricultural pest Ceratitis capitata (Mediterranean fruit fly, or Medfly), we identified a Y-linked gene, Maleness-on-the-Y (MoY), encoding a small protein that is necessary and sufficient for male development. Silencing or disruption of MoY in XY embryos causes feminization, whereas overexpression of MoY in XX embryos induces masculinization. Crosses between transformed XY females and XX males give rise to males and females, indicating that a Y chromosome can be transmitted by XY females. MoY is Y-linked and functionally conserved in other species of the Tephritidae family, highlighting its potential to serve as a tool for developing more effective control strategies against these major agricultural insect pests.


Assuntos
Ceratitis capitata/genética , Genes Ligados ao Cromossomo Y , Processos de Determinação Sexual , Cromossomo Y/genética , Animais , Sequência Conservada , Embrião não Mamífero , Feminino , Genes de Insetos , Masculino , Interferência de RNA
16.
Biol Open ; 8(1)2019 Jan 03.
Artigo em Inglês | MEDLINE | ID: mdl-30498016

RESUMO

A first generation of CRISPR-based gene drives has now been tested in the laboratory in a number of organisms, including malaria vector mosquitoes. Challenges for their use in the area-wide genetic control of vector-borne disease have been identified, including the development of target site resistance, their long-term efficacy in the field, their molecular complexity, and practical and legal limitations for field testing of both gene drive and coupled anti-pathogen traits. We have evaluated theoretically the concept of integral gene drive (IGD) as an alternative paradigm for population replacement. IGDs incorporate a minimal set of molecular components, including drive and anti-pathogen effector elements directly embedded within endogenous genes - an arrangement that in theory allows targeting functionally conserved coding sequences without disrupting their function. Autonomous and non-autonomous IGD strains could be generated, optimized, regulated and imported independently. We performed quantitative modeling comparing IGDs with classical replacement drives and show that selection for the function of the hijacked host gene can significantly reduce the establishment of resistant alleles in the population, while drive occurring at multiple genomic loci prolongs the duration of transmission blockage in the face of pre-existing target site variation. IGD thus has potential as a more durable and flexible population replacement strategy.

17.
Parasit Vectors ; 11(Suppl 2): 654, 2018 Dec 24.
Artigo em Inglês | MEDLINE | ID: mdl-30583720

RESUMO

Major efforts are currently underway to develop novel, complementary methods to combat mosquito-borne diseases. Mosquito genetic control strategies (GCSs) have become an increasingly important area of research on account of their species-specificity, track record in targeting agricultural insect pests, and their environmentally non-polluting nature. A number of programs targeting Aedes and Anopheles mosquitoes, vectors of human arboviruses and malaria respectively, are currently being developed or deployed in many parts of the world. Operationally implementing these technologies on a large scale however, beyond proof-of-concept pilot programs, is hampered by the absence of adequate sex separation methods. Sex separation eliminates females in the laboratory from male mosquitoes prior to release. Despite the need for sex separation for the control of mosquitoes, there have been limited efforts in recent years in developing systems that are fit-for-purpose. In this special issue of Parasites and Vectors we report on the progress of the global Coordinated Research Program on "Exploring genetic, molecular, mechanical and behavioural methods for sex separation in mosquitoes" that is led by the Insect Pest Control Subprogramme of the Joint FAO/IAEA Division of Nuclear Techniques in Food and Agriculture with the specific aim of building efficient sex separation systems for mosquito species. In an effort to overcome current barriers we briefly highlight what we believe are the three main reasons why progress has been so slow in developing appropriate sex separation systems: the availability of methods that are not scalable, the difficulty of building the ideal genetic systems and, finally, the lack of research efforts in this area.


Assuntos
Aedes/genética , Anopheles/genética , Malária/prevenção & controle , Controle de Mosquitos , Mosquitos Vetores/genética , Aedes/fisiologia , Animais , Anopheles/fisiologia , Feminino , Tecnologia de Impulso Genético , Humanos , Infertilidade , Malária/transmissão , Masculino , Mosquitos Vetores/fisiologia , Análise para Determinação do Sexo
18.
Sci Rep ; 8(1): 13125, 2018 09 03.
Artigo em Inglês | MEDLINE | ID: mdl-30177778

RESUMO

The ability to erect rationally-engineered reproductive barriers in animal or plant species promises to enable a number of biotechnological applications such as the creation of genetic firewalls, the containment of gene drives or novel population replacement and suppression strategies for genetic control. However, to date no experimental data exist that explores this concept in a multicellular organism. Here we examine the requirements for building artificial reproductive barriers in the metazoan model Drosophila melanogaster by combining CRISPR-based genome editing and transcriptional transactivation (CRISPRa) of the same loci. We directed 13 single guide RNAs (sgRNAs) to the promoters of 7 evolutionary conserved genes and used 11 drivers to conduct a misactivation screen. We identify dominant-lethal activators of the eve locus and find that they disrupt development by strongly activating eve outside its native spatio-temporal context. We employ the same set of sgRNAs to isolate, by genome editing, protective INDELs that render these loci resistant to transactivation without interfering with target gene function. When these sets of genetic components are combined we find that complete synthetic lethality, a prerequisite for most applications, is achievable using this approach. However, our results suggest a steep trade-off between the level and scope of dCas9 expression, the degree of genetic isolation achievable and the resulting impact on fly fitness. The genetic engineering strategy we present here allows the creation of single or multiple reproductive barriers and could be applied to other multicellular organisms such as disease vectors or transgenic organisms of economic importance.


Assuntos
Sistemas CRISPR-Cas , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Edição de Genes/métodos , Genes de Insetos , Genoma de Inseto , Proteínas de Homeodomínio/genética , Fatores de Transcrição/genética , Animais , Sequência de Bases , Proteína 9 Associada à CRISPR/genética , Proteína 9 Associada à CRISPR/metabolismo , Feminino , Genes Letais , Aptidão Genética , Loci Gênicos , Mutação INDEL , Masculino , Controle da População/métodos , Regiões Promotoras Genéticas , RNA Guia de Cinetoplastídeos/genética , RNA Guia de Cinetoplastídeos/metabolismo , Isolamento Reprodutivo , Alinhamento de Sequência , Ativação Transcricional
19.
CRISPR J ; 1(1): 88-98, 2018 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-30627701

RESUMO

CRISPR-based synthetic sex ratio distorters, which operate by shredding the X-chromosome during male meiosis, are promising tools for the area-wide control of harmful insect pest or disease vector species. X-shredders have been proposed as tools to suppress insect populations by biasing the sex ratio of the wild population toward males, thus reducing its natural reproductive potential. However, to build synthetic X-shredders based on CRISPR, the selection of gRNA targets, in the form of high-copy sequence repeats on the X chromosome of a given species, is difficult, since such repeats are not accurately resolved in genome assemblies and cannot be assigned to chromosomes with confidence. We have therefore developed the redkmer computational pipeline, designed to identify short and highly abundant sequence elements occurring uniquely on the X chromosome. Redkmer was designed to use as input minimally processed whole genome sequence data from males and females. We tested redkmer with short- and long-read whole genome sequence data of Anopheles gambiae, the major vector of human malaria, in which the X-shredding paradigm was originally developed. Redkmer established long reads as chromosomal proxies with excellent correlation to the genome assembly and used them to rank X-candidate kmers for their level of X-specificity and abundance. Among these, a high-confidence set of 25-mers was identified, many belonging to previously known X-chromosome repeats of Anopheles gambiae, including the ribosomal gene array and the selfish elements harbored within it. Data from a control strain, in which these repeats are shared with the Y chromosome, confirmed the elimination of these kmers during filtering. Finally, we show that redkmer output can be linked directly to gRNA selection and off-target prediction. In addition, the output of redkmer, including the prediction of chromosomal origin of single-molecule long reads and chromosome specific kmers, could also be used for the characterization of other biologically relevant sex chromosome sequences, a task that is frequently hampered by the repetitiveness of sex chromosome sequence content.

20.
Genetics ; 207(2): 729-740, 2017 10.
Artigo em Inglês | MEDLINE | ID: mdl-28860320

RESUMO

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.


Assuntos
Anopheles/genética , Cromossomos de Insetos/genética , Evolução Molecular , Hibridização Genética , Mosquitos Vetores/genética , Cromossomo Y/genética , Animais , Fluxo Gênico , Transferência Genética Horizontal , Patrimônio Genético , Aptidão Genética , Infertilidade Masculina/genética , Masculino
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