Publications
62 results found
Meccariello A, 2024, Gene drive and genetic sex conversion in theglobal agricultural pest Ceratitis capitata, Nature Communications, Vol: 15, ISSN: 2041-1723
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.
Taitingfong RI, Triplett C, Vasquez VN, et al., 2022, Exploring the value of a global gene drive project registry, NATURE BIOTECHNOLOGY, ISSN: 1087-0156
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- Citations: 1
Habtewold T, Tapanelli S, Masters EKG, et al., 2022, The circadian clock modulates <i>Anopheles gambiae</i> infection with <i>Plasmodium falciparum</i>, PLOS ONE, Vol: 17, ISSN: 1932-6203
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- Citations: 2
Windbichler N, 2022, Gene drive mosquitoes can aid malaria elimination by retarding Plasmodium sporogonic development, Science Advances, Vol: 8, Pages: 1-9, ISSN: 2375-2548
Abstract: Gene drives hold promise for the genetic control of malaria vectors. The development of vector population modification strategies hinges on the availability of effector mechanisms impeding parasite development in transgenic mosquitoes. We augmented a midgut gene of the malaria mosquito Anopheles gambiae to secrete two exogenous antimicrobial peptides, Magainin 2 and Melittin. This small genetic modification, capable of efficient non-autonomous gene drive, hampers oocyst development in both Plasmodium falciparum and Plasmodium berghei. It delays the release of infectious sporozoites while it simultaneously reduces the lifespan of homozygous female transgenic mosquitoes. Modeling the spread of this modification using a large-scale agent-based model of malaria epidemiology reveals that it can break the cycle of disease transmission across a range of transmission intensities.
Leung S, Windbichler N, Wenger EA, et al., 2022, Population replacement gene drive characteristics for malaria elimination in a range of seasonal transmission settings: a modelling study, MALARIA JOURNAL, Vol: 21
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- Citations: 4
Ellis D, Avraam GP, Hoermann A, et al., 2022, Testing non-autonomous antimalarial gene drive effectors using self-eliminating drivers in the African mosquito vector <i>Anopheles gambiae</i>, PLOS GENETICS, Vol: 18, ISSN: 1553-7404
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- Citations: 3
Nash A, Paolo C, Hoermann A, et al., 2022, Intronic gRNAs for the construction of minimal gene drive systems, Frontiers in Bioengineering and Biotechnology, Vol: 10, Pages: 1-10, ISSN: 2296-4185
Gene drives are promising tools for the genetic control of insect vector or pest populations. CRISPR-based gene drives are generally highly complex synthetic constructs consisting of multiple transgenes and their respective regulatory elements. This complicates the generation of new gene drives and the testing of the behavior of their constituent functional modules. Here, we explored the minimal genetic components needed to constitute autonomous gene drives in Drosophila melanogaster. We first designed intronic gRNAs that can be located directly within coding transgene sequences and tested their functions in cell lines. We then integrated a Cas9 open reading frame hosting such an intronic gRNA within the Drosophila rcd-1r locus that drives the expression in the male and female germlines. We showed that upon removal of the fluorescent transformation marker, the rcd-1rd allele supports efficient gene drive. We assessed the propensity of this driver, designed to be neutral with regards to fitness and host gene function, to propagate in caged fly populations. Because of their simplicity, such integral gene drives could enable the modularization of drive and effector functions. We also discussed the possible biosafety implications of minimal and possibly recoded gene drives.
Hoermann A, Habtewold T, Selvaraj P, et al., 2022, Gene Drive Mosquitoes Can Aid Malaria Elimination by Retarding Plasmodium Sporogonic Development
<jats:title>Abstract</jats:title><jats:p>Gene drives hold promise for the genetic control of malaria vectors. The development of vector population modification strategies hinges on the availability of effector mechanisms impeding parasite development in transgenic mosquitoes. We augmented a midgut gene of the malaria mosquito <jats:italic>Anopheles gambiae</jats:italic> to secrete two exogenous antimicrobial peptides, Magainin 2 and Melittin. This small genetic modification, capable of efficient non-autonomous gene drive, hampers oocyst development in both <jats:italic>Plasmodium falciparum</jats:italic> and <jats:italic>Plasmodium berghei</jats:italic>. It delays the release of infectious sporozoites while it simultaneously reduces the lifespan of homozygous female transgenic mosquitoes. Modeling the spread of this modification using a large-scale agent-based model of malaria epidemiology reveals that it can break the cycle of disease transmission across a range of endemic settings.</jats:p><jats:sec><jats:title>One sentence summary</jats:title><jats:p>We developed a gene drive effector that retards <jats:italic>Plasmodium</jats:italic> development in transgenic <jats:italic>Anopheles gambiae</jats:italic> mosquitoes via the expression of antimicrobial peptides in the midgut and which is predicted to eliminate malaria under a range of transmission scenarios.</jats:p></jats:sec>
Tapanelli S, Inghilterra MG, Cai J, et al., 2021, Assessment of <i>Plasmodium falciparum</i> infection and fitness of genetically modified <i>Anopheles gambiae</i> aimed at mosquito population replacement
<jats:title>Abstract</jats:title><jats:p>Genetically modified (GM) mosquitoes expressing anti-plasmodial effectors propagating through wild mosquito populations by means of gene drive is a promising tool to support current malaria control strategies. The process of generating GM mosquitoes involves genetic transformation of mosquitoes from a laboratory colony and, often, interbreeding with other GM lines to cross in auxiliary traits. These mosquito colonies and GM lines thus often have different genetic backgrounds and GM lines are invariably highly inbred, which in conjunction with their independent rearing in the laboratory may translate to differences in their susceptibility to malaria parasite infection and life history traits. Here, we show that laboratory <jats:italic>Anopheles gambiae</jats:italic> colonies and GM lines expressing Cas9 and Cre recombinase vary greatly in their susceptibility to <jats:italic>Plasmodium falciparum</jats:italic> NF54 infection. Therefore, the choice of mosquitoes to be used as a reference when conducting infection or life history trait assays requires careful consideration. To address these issues, we established an experimental pipeline involving genetic crosses and genotyping of mosquitoes reared in shared containers throughout their lifecycle. We used this protocol to examine whether GM lines expressing the antimicrobial peptide (AMP) Scorpine in the mosquito midgut interfere with parasite infection and mosquito survival. We demonstrate that Scorpine expression in the Peritrophin 1 (Aper1) genomic locus reduces both <jats:italic>P</jats:italic>. <jats:italic>falciparum</jats:italic> sporozoite prevalence and mosquito lifespan; both these phenotypes are likely to be associated with the disturbance of the midgut microbiota homeostasis. These data lead us to conclude that the Aper1-Sco GM line could be used in proof-of-concept experiments aimed at mosquito populat
Meccariello A, Krsticevic F, Colonna R, et al., 2021, Engineered sex ratio distortion by X-shredding in the global agricultural pest <i>Ceratitis capitata</i>, BMC BIOLOGY, Vol: 19
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- Citations: 18
Hoermann A, Tapanelli S, Capriotti P, et al., 2021, Converting endogenous genes of the malaria mosquito into simple non-autonomous gene drives for pope ion replacement, eLife, Vol: 10, Pages: 1-22, ISSN: 2050-084X
Gene drives for mosquito population replacement are promising tools for malaria control. However, there is currently no clear pathway for safely testing such tools in endemic countries. The lack of well-characterized promoters for infection-relevant tissues and regulatory hurdles are further obstacles for their design and use. Here we explore how minimal genetic modifications of endogenous mosquito genes can convert them directly into non-autonomous gene drives without disrupting their expression. We co-opted the native regulatory sequences of three midgut-specific loci of the malaria vector Anopheles gambiae to host a prototypical antimalarial molecule and guide-RNAs encoded within artificial introns that support efficient gene drive. We assess the propensity of these modifications to interfere with the development of Plasmodium falciparum and their effect on fitness. Because of their inherent simplicity and passive mode of drive such traits could form part of an acceptable testing pathway of gene drives for malaria eradication.
Habtewold T, Sharma A, Wyer C, et al., 2021, Plasmodium oocysts respond with dormancy to crowding and nutritional stress, Scientific Reports, Vol: 11, ISSN: 2045-2322
Malaria parasites develop as oocysts in the mosquito for several days before they are able to infect a human host. During this time, mosquitoes take bloodmeals to replenish their nutrient and energy reserves needed for flight and reproduction. We hypothesized that these bloodmeals are critical for oocyst growth and that experimental infection protocols, typically involving a single bloodmeal at the time of infection, cause nutritional stress to the developing oocysts. Therefore, enumerating oocysts disregarding their growth and differentiation state may lead to erroneous conclusions about the efficacy of transmission blocking interventions. Here, we examine this hypothesis in Anopheles coluzzii mosquitoes infected with the human and rodent parasites Plasmodium falciparum and Plasmodium berghei, respectively. We show that oocyst growth and maturation rates decrease at late developmental stages as infection intensities increase; an effect exacerbated at very high infection intensities but fully restored with post infection bloodmeals. High infection intensities and starvation conditions reduce RNA Polymerase III activity in oocysts unless supplemental bloodmeals are provided. Our results suggest that oocysts respond to crowding and nutritional stress with a dormancy-like strategy, which urges the development of alternative methods to assess the efficacy of transmission blocking interventions.
Price TAR, Windbichler N, Unckless RL, et al., 2020, Resistance to natural and synthetic gene drive systems, JOURNAL OF EVOLUTIONARY BIOLOGY, Vol: 33, Pages: 1345-1360, ISSN: 1010-061X
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- Citations: 28
Meccariello A, Krsticevic F, Colonna R, et al., 2020, Engineered sex distortion in the global agricultural pest<i>Ceratitis capitata</i>
<jats:title>Abstract</jats:title><jats:p>Genetic sex ratio distorters have potential for the area-wide control of harmful insect populations. Endonucleases targeting the X-chromosome and whose activity is restricted to male gametogenesis have recently been pioneered as a means to engineer such traits. Here we enabled endogenous CRISPR/Cas9 and CRISPR/Cas12a activity during spermatogenesis of the Mediterranean fruit fly<jats:italic>Ceratitis capitata</jats:italic>, 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 they yielded a significant and consistent distortion of the sex ratio in independent transgenic strains and a combination of distorters induced a strong bias towards males (~80%). Our results demonstrate the design of sex distorters in a non-model organism and suggest that strains with characteristics suitable for field application could be developed for a range of medically or agriculturally relevant insect species.</jats:p>
Selvaraj P, Wenger EA, Bridenbecker D, et al., 2020, Vector genetics, insecticide resistance and gene drives: An agent-based modeling approach to evaluate malaria transmission and elimination, PLOS COMPUTATIONAL BIOLOGY, Vol: 16, ISSN: 1553-734X
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- Citations: 11
Hoermann A, Tapanelli S, Capriotti P, et al., 2020, Converting endogenous genes of the malaria mosquito into simple non-autonomous gene drives for population replacement
<jats:title>Abstract</jats:title><jats:p>Gene drives for mosquito population replacement are promising tools for malaria control. However, there is currently no clear pathway for safely testing such tools in endemic countries. The lack of well-characterized promoters for infection-relevant tissues and regulatory hurdles are further obstacles for their design and use. Here we explore how minimal genetic modifications of endogenous mosquito genes can convert them directly into non-autonomous gene drives without disrupting their expression. We co-opted the native regulatory sequences of three midgut-specific loci of the malaria vector <jats:italic>Anopheles gambiae</jats:italic> to host a prototypical antimalarial molecule and guide-RNAs encoded within artificial introns, that support efficient gene drive. We assess the propensity of these modifications to interfere with the development of <jats:italic>Plasmodium falciparum</jats:italic> and their effect on fitness. Because of their inherent simplicity and passive mode of drive such traits could form part of an accepted testing pathway of gene drives for malaria eradication.</jats:p>
Redhai S, Pilgrim C, Gaspar P, et al., 2020, An intestinal zinc sensor regulates food intake and developmental growth, Nature, Vol: 580, Pages: 263-268, ISSN: 0028-0836
In cells, organs and whole organisms, nutrient sensing is key to maintaining homeostasis and adapting to a fluctuating environment1. In many animals, nutrient sensors are found within the enteroendocrine cells of the digestive system; however, less is known about nutrient sensing in their cellular siblings, the absorptive enterocytes1. Here we use a genetic screen in Drosophila melanogaster to identify Hodor, an ionotropic receptor in enterocytes that sustains larval development, particularly in nutrient-scarce conditions. Experiments in Xenopus oocytes and flies indicate that Hodor is a pH-sensitive, zinc-gated chloride channel that mediates a previously unrecognized dietary preference for zinc. Hodor controls systemic growth from a subset of enterocytes—interstitial cells—by promoting food intake and insulin/IGF signalling. Although Hodor sustains gut luminal acidity and restrains microbial loads, its effect on systemic growth results from the modulation of Tor signalling and lysosomal homeostasis within interstitial cells. Hodor-like genes are insect-specific, and may represent targets for the control of disease vectors. Indeed, CRISPR–Cas9 genome editing revealed that the single hodor orthologue in Anopheles gambiae is an essential gene. Our findings highlight the need to consider the instructive contributions of metals—and, more generally, micronutrients—to energy homeostasis.
Habtewold T, Sharma A, Wyer CAS, et al., 2020, Plasmodium oocysts respond with dormancy to crowding and nutritional stress, Publisher: Cold Spring Harbor Laboratory
Malaria parasites develop and grow as oocysts in the mosquito for several days before being able to infect another human. During this time, mosquitoes take regular bloodmeals to replenish their nutrient and energy reserves needed for flight and reproduction. We hypothesized that supplemental bloodmeals are critical for oocyst growth and that experimental infection protocols, typically involving a single bloodmeal, cause nutritional stress to developing oocysts. Therefore, enumerating oocysts independently of their growth and differentiation state may lead to erroneous conclusions regarding the efficacy of malaria transmission blocking interventions. We tested this hypothesis in Anopheles coluzzii mosquitoes infected with human and rodent parasites Plasmodium falciparum and Plasmodium berghei , respectively. We find that oocyst growth rates decrease at late developmental stages as infection intensities increase; an effect exacerbated at very high infection intensities. Oocyst growth and differentiation can be restored by supplemental bloodmeals even at high infection intensities. We show that high infection intensities as well as starvation conditions reduce RNA Polymerase III activity in oocysts unless supplemental bloodmeals are provided. Our data suggest that oocysts respond to crowding and nutritional stress by employing a dormancy-like strategy and urge development of alternative methods to assess the efficacy of transmission blocking interventions.
Fasulo B, Meccariello A, Morgan M, et al., 2020, A fly model establishes distinct mechanisms for synthetic CRISPR/Cas9 sex distorters, PLOS GENETICS, Vol: 16, ISSN: 1553-7404
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- Citations: 24
Fasulo B, Meccariello A, Papathanos PA, et al., 2020, SYNTHETIC SEX RATIO DISTORTERS BASED ON CRISPR FOR THE CONTROL OF HARMFUL INSECT POPULATIONS, AREA-WIDE INTEGRATED PEST MANAGEMENT, Editors: Hendrichs, Pereira, Vreysen, Publisher: CRC PRESS-TAYLOR & FRANCIS GROUP, Pages: 843-855, ISBN: 978-0-367-76986-4
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- Citations: 1
Fasulo B, Meccariello A, Morgan M, et al., 2019, A fly model establishes distinct mechanisms for synthetic CRISPR/Cas9 sex distorters
<jats:p>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<jats:italic>Drosophila</jats:italic>models for two distinct mechanisms for CRISPR/Cas9 sex-ratio distortion - “X-shredding” and “X-meddling” - and dissected their target-site requirements and repair dynamics. X-shredding relies on sufficient meiotic activity of the endonuclease to overpower DNA repair and can operate on a single repeat cluster of non-essential sequences. X-meddling by contrast, i.e. targeting putative haplolethal X-linked genes, induced a bias towards males that is coupled to a loss in reproductive output, although a dominant-negative effect may drive the mechanism of female lethality. Our model system will guide the study and the application of sex distorters to medically or agriculturally important insect target species.</jats:p>
Dahalan FA, Churcher TS, Windbichler N, et al., 2019, The male mosquito contribution towards malaria transmission: Mating influences the Anopheles female midgut transcriptome and increases female susceptibility to human malaria parasites., PLoS Pathogens, Vol: 15, Pages: 1-19, ISSN: 1553-7366
Mating causes dramatic changes in female physiology, behaviour, and immunity in many insects, inducing oogenesis, oviposition, and refractoriness to further mating. Females from the Anopheles gambiae species complex typically mate only once in their lifetime during which they receive sperm and seminal fluid proteins as well as a mating plug that contains the steroid hormone 20-hydroxyecdysone. This hormone, which is also induced by blood-feeding, plays a major role in activating vitellogenesis for egg production. Here we show that female Anopheles coluzzii susceptibility to Plasmodium falciparum infection is significantly higher in mated females compared to virgins. We also find that mating status has a major impact on the midgut transcriptome, detectable only under sugar-fed conditions: once females have blood-fed, the transcriptional changes that are induced by mating are likely masked by the widespread effects of blood-feeding on gene expression. To determine whether increased susceptibility to parasites could be driven by the additional 20E that mated females receive from males, we mimicked mating by injecting virgin females with 20E, finding that these females are significantly more susceptible to human malaria parasites than virgin females injected with the control 20E carrier. Further RNAseq was carried out to examine whether the genes that change upon 20E injection in the midgut are similar to those that change upon mating. We find that 79 midgut-expressed genes are regulated in common by both mating and 20E, and 96% (n = 76) of these are regulated in the same direction (up vs down in 20E/mated). Together, these findings show that male Anopheles mosquitoes induce changes in the female midgut that can affect female susceptibility to P. falciparum. This implies that in nature, males might contribute to malaria transmission in previously unappreciated ways, and that vector control strategies that target males may have additional benefits towards reducing transm
Giese B, Friess JL, Barton NH, et al., 2019, Gene Drives: Dynamics and Regulatory Matters-A Report from the Workshop "Evaluation of Spatial and Temporal Control of Gene Drives," April 4-5, 2019, Vienna, BIOESSAYS, Vol: 41, ISSN: 0265-9247
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- Citations: 1
Meccariello A, Salvemini M, Primo P, et al., 2019, Maleness-on-the-Y (MoY) orchestrates male sex determination in major agricultural fruit fly pests, Sciecnce, Vol: 365, Pages: 1457-1460, ISSN: 0036-8075
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.
Habtewold T, Tapanelli S, Ellen KG M, et al., 2019, Streamlined SMFA and mosquito dark-feeding regime significantly improve malaria transmission-blocking assay robustness and sensitivity, Malaria Journal, Vol: 18, ISSN: 1475-2875
BackgroundThe development of malaria transmission-blocking strategies including the generation of malaria refractory mosquitoes to replace the wild populations through means of gene drives hold great promise. The standard membrane feeding assay (SMFA) that involves mosquito feeding on parasitized blood through an artificial membrane system is a vital tool for evaluating the efficacy of transmission-blocking interventions. However, despite the availability of several published protocols, the SMFA remains highly variable and broadly insensitive.MethodsThe SMFA protocol was optimized through coordinated culturing of Anopheles coluzzii mosquitoes and Plasmodium falciparum parasite coupled with placing mosquitoes under a strict dark regime before, during, and after the gametocyte feed.ResultsA detailed description of essential steps is provided toward synchronized generation of highly fit An. coluzzii mosquitoes and P. falciparum gametocytes in preparation for an SMFA. A dark-infection regime that emulates the natural vector-parasite interaction system is described, which results in a significant increase in the infection intensity and prevalence. Using this optimal SMFA pipeline, a series of putative transmission-blocking antimicrobial peptides (AMPs) were screened, confirming that melittin and magainin can interfere with P. falciparum development in the vector.ConclusionA robust SMFA protocol that enhances the evaluation of interventions targeting human malaria transmission in laboratory setting is reported. Melittin and magainin are identified as highly potent antiparasitic AMPs that can be used for the generation of refractory Anopheles gambiae mosquitoes.
Nash A, Urdaneta Mignini G, Beaghton A, et al., 2019, Integral Gene Drives for population replacement, Biology Open, Vol: 8, ISSN: 2046-6390
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.
Waters AJ, Capriotti P, Gaboriau DCA, et al., 2018, Rationally-engineered reproductive barriers using CRISPR & CRISPRa: an evaluation of the synthetic species concept in Drosophila melanogaster, Scientific Reports, Vol: 8, ISSN: 2045-2322
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.
Nash A, Urdaneta GM, K Beaghton A, et al., 2018, Integral Gene Drives: an “operating system” for population replacement
<jats:title>Abstract</jats:title><jats:p>First generation CRISPR-based gene drives have now been tested in the laboratory in a number of organisms including malaria vector mosquitoes. A number of challenges for their use in the area-wide genetic control of vector-borne disease have been identified. These include the development of target site resistance, their long-term efficacy in the field, their molecular complexity, and the practical and legal limitations for field testing of both gene drive and coupled anti-pathogen traits. To address these challenges, we have evaluated the concept of Integral Gene Drive (IGD) as an alternative paradigm for population replacement. IGDs incorporate a minimal set of molecular components, including both the drive and the anti-pathogen effector elements directly embedded within endogenous genes – an arrangement which we refer to as gene “hijacking”. This design would allow autonomous and non-autonomous IGD traits and strains to be generated, tested, optimized, regulated and imported independently. We performed quantitative modelling 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 hedging drive over multiple genomic loci prolongs the duration of transmission blockage in the face of pre-existing target-site variation. IGD thus has the potential to yield more durable and flexible population replacement traits.</jats:p>
Waters AJ, Capriotti P, Gaboriau D, et al., 2018, Rationally-engineered reproductive barriers using CRISPR & CRISPRa: an evaluation of the synthetic species concept inDrosophila melanogaster, Publisher: bioRxiv
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 miss-activation 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.
Papathanos PA, Windbichler N, 2018, Redkmer: An assembly-free pipeline for the identification of abundant and specific X-chromosome target sequences for X-shredding by CRISPR endonucleases, The Crispr Journal, Vol: 1, Pages: 88-98, ISSN: 2573-1599
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 charact
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