modelingFull text
Molecular biology and evolution · 2026-Jul-02 · PMID 42488959
Prateek Verma, Omar S Akbari, John M Marshall
Invasive rodents are major contributors to biodiversity loss, particularly on islands where native species have evolved in their absence. Genetic biocontrol offers a promising suite of species-specific technologies that may contribute to rodent suppression and elimination. Here, we evaluate the potential of Y-linked genome editors (YLEs), which contain a nuclease that disrupts a female-essential fertility or viability gene, to suppress or eliminate a mouse population on a small, isolated island. We use the MouseGD simulation framework to model the inheritance patterns of YLEs and other genetic biocontrol tools in the context of rodent life history, calculating elimination probabilities, times to elimination and other outcomes for a range of construct and release parameters. Simulations suggest that YLEs are more efficient than other self-limiting tools, and are capable of achieving elimination within a short timeframe for modest release sizes. For an in silico mouse population size of 10,000, elimination is simulated within 5 years for releases of 350 males per month, and within 10 years for releases of 150 males per month. This scale of production is within existing capabilities, potentially enabling suppression to encompass a larger spatial scale. Simulations suggest that elimination could be achieved for YLEs targeting both haploinsufficient and haplosufficient target genes. Gene drives were simulated to achieve suppression and elimination for smaller release sizes; but also to spread to non-target populations. In contrast, YLEs display minimal spillover. These characteristics present YLEs as a promising, ecologically manageable biocontrol tool for elimination of invasive rodents and conservation of native species.
Gene drivesGene drive
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Journal of medical entomology · 2026-Jan-20 · PMID 41144297
Eddie Hill, Maria J Della Rocca, Robyn Raban, Nicolas Herard, Antonio Esho, Monique S Sun, Rhodri T M Edwards, Nicholas Boechler, Omar S Akbari
Mosquito-borne disease and nuisance biting from mosquitoes have severe health and economic consequences. Conventional fabrics are typically not effective at providing protection against mosquito bites, and fabrics treated with repellents and/or insecticides are limited by rising insecticide resistance, risk of significant dermatologic and neurologic side effects, and decreased efficacy with washing and time. The goal of this study was to identify commercially available, repellent/insecticide-free, comfortable fabrics that block bites from three genera of mosquitoes that are known to transmit dangerous infectious diseases with widespread distribution: Aedes, Anopheles, and Culex. To do this, we evaluated fabrics from Ripstop By the Roll LLC in a step-wise series of mouse blood-feeding and behavioral bioassays. Out of 88 fabrics, 53 were found to be blood-feed-proof. These fabrics were more likely to have a higher areal weight density (AWD) and a polyurethane coating than blood-feed-susceptible fabrics. Of the six most comfortable fabrics by subjective hand-feel testing, five were definitively bite-proof during behavioral bioassays. These five fabrics varied substantially in AWD, thickness, finish/coating, and fiber pattern. None of them had a polyurethane coating. Three of them were breathable, making them appropriate for active-wear clothing. Overall, the bite-proof fabrics identified in this study have the potential to significantly reduce mosquito biting and the transmission of mosquito-borne diseases.
AnophelesCulexMosquitoes
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Journal of molecular biology · 2026-Jan-15 · PMID 41317788
Daniel J Brogan, Calvin P Lin, Elena Dalla Benetta, Tianqi Wang, Fangying Chen, Harry Li, Claire Lin, Elizabeth A Komives, Omar S Akbari
The recent discovery of the type III-E class of CRISPR-Cas effectors has reshaped our fundamental understanding of CRISPR-Cas evolution and classification. Type III-E effectors are composed of several Cas7-like domains and a single Cas11-like domain naturally fused together to create a single polypeptide capable of targeting and degrading RNA. Here we identified a novel type III-E-like effector composed of three Cas7 domains and a Cas1 domain which was not active but could be engineered into an active chimeric RNA-targeting Cas effector by domain additions and swaps from other type III-E effectors. The results reveal that various domains in type III-E effectors can be swapped for the equivalent domain from a different type III-E effector. Remarkably, the Cas1 domain located at the C-terminus of Cas7-1 could be swapped in place of the Cas11 domain located between the Cas7.1 and the Cas7.2 domains of DiCas7-11. The results reveal a new modality for engineering type III-E effectors from the blueprints found in nature.
CRISPR
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Nature communications · 2026-Feb-20 · PMID 41720789
Yuri Vainer, Evyatar Sar-Shalom, Yinliang Wang, Robert M Huff, Dor Perets, Esther Yakir, Majid Ghaninia, Iliano V Coutinho-Abreu, Carlos Ruiz, Dhivya Rajamanickam, Alon Warburg, Omar S Akbari, Philippos A Papathanos, Rickard Ignell, Jeffrey A Riffell, Ronald Jason Pitts, Jonathan D Bohbot
Plant-derived repellents containing borneol and camphor have been used for centuries, yet the sensory mechanisms that mediate their effects in mosquitoes have remained obscure. Here, we show that culicine mosquitoes possess a dedicated olfactory pathway for detecting bicyclic monoterpenoids. We identify odorant receptor 49 (OR49) as a conserved and highly selective borneol receptor expressed in a distinct neuron of the maxillary palp. This neuron forms a defined projection to the MD3 glomerulus in the antennal lobe, establishing a labeled line for plant-derived repellents. Genetic disruption of Or49 abolishes electrophysiological responses to borneol and markedly diminishes avoidance behavior during human host-seeking. These results reveal that female mosquitoes detect borneol through a specialized sensory channel distinct from those mediating attraction to human cues. Our findings provide a mechanistic framework for understanding how plant volatiles shape mosquito behavior and offer a foundation for the rational design of next-generation olfactory repellents.
Mosquito neurobiologyMosquitoes
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Communications biology · 2026-Feb-05 · PMID 41644661
Serafima Davydova, Junru Liu, Nikolay P Kandul, Igor Antoshechkin, Jonathan Mann, W Evan Braswell, Omar S Akbari, Angela Meccariello
Insect pest population control via sterile insect technique markedly benefits from separation by sex prior to release. To simplify this process, traditional genetics has been deployed to develop genetic sexing strains (GSSs) for several disease vectors and agricultural pests of vast economic significance, although very few are applied in the field due to associated fitness costs and instability. In this study, we generated a method to engineer cisgenic GSS (CGSS) in insects. We use CRISPR/Cas9-mediated homology-directed repair to seamlessly translocate a sex-specific alternatively spliced intron into a dominant phenotypic gene generating a genetically stable strain that enables sex-sorting by eye. To achieve this feat, we use Ceratitis capitata as our model and relied on the sex-specifically spliced intron of its endogenous transformer gene, which we seamlessly inserted a copy into the pupal colouration white pupae gene. This minimal modification resulted in the generation of a homozygous strain we term IMPERIAL that was genetically and phenotypically stable where all female pupae are brown while male pupae are white with overall good fitness. By minimally editing the genome, our novel CGSS approach can be applied to other pests that may aid more efficient and economically suitable pest control.
Sterile insect techniquesSex determinationVector controlCRISPRSIT
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Current opinion in insect science · 2026-Aug-13 · PMID 41833899
Yoosook Lee, Omar S Akbari
No abstract is available from PubMed.
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G3 (Bethesda, Md.) · 2026-Aug-05 · PMID 41706533
Michael S Overton, Sean E Guy, Xingsen Chen, Alena Martsul, Krypton Carolino, Omar S Akbari, Justin R Meyer, Sergey Kryazhimskiy
Homing-based CRISPR-Cas9 gene drives (CCGDs) are powerful tools for genetic control of wild populations, with applications from disease eradication to species conservation. However, Cas9 alone and in a complex with guide RNA can cause double-stranded DNA breaks at off-target sites, which could increase the mutational load and lead to unintended loss-of-heterozygosity (LOH) events. These undesired effects raise potential concerns about the long-term evolutionary safety of CCGDs, but the magnitude of these effects is unknown. To measure how the presence of a CCGD or a Cas9 alone in the genome affects the rates of LOH events and de novo mutations, we carried out a mutation accumulation experiment in yeast Saccharomyces cerevisiae. We found no detectable effects on the genome-wide rates of mutations or LOH events. Our power calculations suggest that CCGD or Cas9 affect these rates by less than 30%, which is much less than natural variation for these traits in yeast. A more detailed examination shows that CCGD or Cas9 may alter the lengths and genomic distributions of LOH events, but the statistical support for these effects is weak. Thus, our results demonstrate that CCGDs impose at most a weak additional mutational burden in the yeast model. Although mutagenic effects of gene drives need to be further evaluated in other systems, our results add credence to the proposition that the evolutionary risks posed by well-designed gene drives may be acceptable.
Gene drivesCRISPRGene drive
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Current opinion in microbiology · 2026-Apr-10 · PMID 41671611
Robyn Raban, Anthony A James, Omar S Akbari
CRISPR-based gene drive (GD) systems bias allele inheritance during meiosis, enabling transgenes to spread at rates exceeding Mendel's law of segregation. This capability underlies their potential as powerful tools for controlling mosquito-borne diseases. GDs can be engineered either to suppress mosquito populations or to modify them by introducing traits that block pathogen transmission. Recent advances have focused on improving evolutionary stability, with modeling studies providing insights into expected population dynamics. With a focus on the most current population modification GDs, we discuss advances in GD architectures - including integral and allelic drives, combined modification-suppression systems, and both homing and non-homing toxin-antidote designs - that expand the range of possible strategies and address limitations of early homing drives. Numerous antipathogen effectors with strong pathogen-blocking activity can now be coupled to these systems, with current efforts assessing their durability against genetically diverse pathogens. Key challenges remain, including resistance evolution, ecological impacts, and long-term stability. Nonetheless, GDs offer a promising approach for reducing disease transmission, especially in regions where conventional interventions are difficult to sustain.
Gene drivesVector controlMosquitoesCRISPRGene drive
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Fly · 2026 · PMID 42307111
Eddie Hill, Robyn Raban, Omar S Akbari
Chemical insecticides have long been used to control agricultural pests, but their widespread application has driven resistance and caused significant ecological and health impacts. CRISPR/Cas9-based genetic biocontrol technologies, including the precision-guided sterile insect technique (pgSIT) and homing gene drives (HGDs), offer targeted alternatives for suppressing pest populations with reduced environmental cost. pgSIT produces sterile males without radiation and achieves high mating competitiveness without multigenerational persistence. In contrast, HGDs bias inheritance to enable sustained population suppression through disruption of essential fertility or viability genes, albeit with greater ecological and regulatory considerations. Experimental applications in multiple agricultural pest species demonstrate robust suppression efficacy. Emerging innovations, including temperature-inducible pgSIT systems, may further streamline mass-rearing and deployment. Together, these approaches have the potential to reduce crop losses and reliance on chemical insecticides while lowering long-term management costs. Their successful integration into agricultural systems will depend on rigorous risk assessment, regulatory oversight, and stakeholder engagement.
Sterile insect techniquesGene drivesVector controlCRISPRGene drivepgSIT
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Scientific African · 2025-Sep-3 · PMID 41018557
William A C Gendron, Robyn Raban, Agastya Mondal, Héctor M Sánchez C, David Zilberman, Patrick G C Ilboudo, Umberto D'Alessandro, John M Marshall, Omar S Akbari
Malaria control has primarily been achieved through vector control, but current methods are insufficient to achieve elimination. Precision guided sterile insect technique (pgSIT) is a mosquito suppression technique that generates sterile male mosquitoes for mass release. Our previous studies showed that this intervention is expected to be highly cost-effective in a malaria endemic region of West Africa, but these estimates used only 15-31% capacity for sex sorting, which is the limiting production step and a primary cost. We, therefore, determined the most cost efficient facility size by calculating the cost per million Anopheles gambiae suppressed as the facility was scaled up to suppress more mosquitoes. We developed an optimized facility size per 9.2 million mosquitoes suppressed, which can be a framework for scaling and increases the cost effectiveness of this intervention. The development of this intervention can potentially interrupt malaria transmission, strengthen local public health institutions, create manufacturing capacity, provide local jobs, and enhance regional health security capabilities that are more resilient to disruptions in supply chains and malaria investment.
Sterile insect techniquesVector controlAnophelesMosquitoespgSITSIT
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Proceedings. Biological sciences · 2025-Oct-29 · PMID 41157878
Melissa Leon Noreña, Sandeep Jandu, John Boyi, Lucia Ibarra Bouzada, Angela Rouyar, Iliano Coutinho Abreu, Fanying Chen, Omar S Akbari, R Jason Pitts, Jeffrey A Riffell
Invasive mosquito species are key vectors of arboviral diseases, like dengue, zika and chikungunya, posing significant public health challenges worldwide. These issues are worsened by urbanization, climate change and insecticide resistance, driving research into new control methods. Adult mosquitoes are attracted to plant nutrient sources essential for flight and reproduction. However, few studies have examined the odours emitted by these sources, and little is known about the olfactory neurobiology of mosquito-plant interactions. This review synthesizes current knowledge on the broad classes of volatile compounds that modulate mosquito behaviour, focusing on the olfactory processes underlying mosquito responses to plant nutrient sources. We also discuss the application of neurogenetic tools for investigating the role of olfactory receptor genes and neural circuits in mosquito ecology. Finally, we explore how insights from these studies can inform and enhance mosquito control strategies, including developing synthetic lures for attractive toxic sugar baits and improved trapping and surveillance technologies. Defining the olfactory receptors, sensory neurons and neural circuits mediating attraction or repellency to plant odours is crucial for optimizing mosquito monitoring and control interventions.
Antiviral engineeringMosquitoes
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Proceedings of the National Academy of Sciences of the United States of America · 2025-Oct-28 · PMID 41129220
Shadi Charara, Jonathan Choy, Kalyani Cauwenberghs, Pawel Vijayakumar, Renny Ng, Keun-Young Kim, Shih-Che Weng, Omar S Akbari, Mark H Ellisman, Scott A Rifkin, Chih-Ying Su
Hematophagous mosquitoes use CO 2 as a key arousal signal that gates behavioral responses to host-derived cues. In Aedes aegypti , CO 2 is detected by olfactory receptor neurons (ORNs) housed in the sensory hairs (sensilla) on the maxillary palp. While the molecular mechanism and behavioral significance of CO 2 sensing have been well studied in mosquitoes, the nanoscale three-dimensional structures of their CO 2 -sensing ORNs and associated cells have remained unclear. Using serial block-face scanning electron microscopy, we characterize the CO 2 -sensing cpA neuron and its odor-sensitive neighbors, cpB and cpC, within the capitate sensilla of A. aegypti. Notably, cpA neurons are significantly larger, with an outer dendritic surface area 8 to 12 times greater than that of cpB and cpC neurons. This expanded CO 2 -sensing surface arises from its unique architecture, consisting of numerous flattened dendritic sheets folded into intricate lamellae. In contrast, cpB and cpC dendrites exhibit sparse, narrow cylindrical branches. Moreover, the cpA axon displays a prominent pearls-on-a-string morphology, with numerous mitochondria-rich, nonsynaptic varicosities connected by thin cables. Remarkably, a glial cell and an auxiliary cell together ensheathe the cpA soma but not cpB or cpC, suggesting a specialized role in supporting cpA function. Compared to Drosophila CO 2 -sensitive ORNs, a larger portion of the cpA outer dendrite is embedded within the sensillum cuticle, potentially improving access to environmental CO 2 . These findings reveal key morphological specializations of cpA neurons, thereby advancing our understanding of mosquito sensory biology and laying the groundwork for future studies on the molecular basis and functional ramifications of these anatomical adaptations.
Mosquito neurobiologyAedes aegyptiDrosophilaMosquitoes
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Scientific reports · 2025-Oct-08 · PMID 41062528
Andrea L Smidler, Eryney A Marrogi, Sean Scott, Enzo Mameli, Daniel Abernathy, Omar S Akbari, George M Church, Flaminia Catteruccia, Kevin Esvelt
Gene drives are selfish genetic elements which promise to be powerful tools in the fight against vector-borne diseases such as malaria. We previously proposed population replacement gene drives designed to better withstand the evolution of resistance by homing through haplolethal loci. Because most mutations in the wild-type allele that would otherwise confer resistance are lethal, only successful drive homing and functional r1 alleles permits the cell to survive. Here we outline the development and characterization of two ΦC31-Recombination mediated cassette exchange gene drive docking lines with these features in Anopheles gambiae, a first step towards construction of robust gene drives in this important malaria vector. We outline adaption of the technique HACK (Homology Assisted CRISPR knockin) to knock-in two docking site sequences into a paired putative haplolethal-haplosufficient (Ribosome-Proteasome) locus, and confirm that these docking lines permit insertion of drive-relevant transgenes. We report the first anopheline proteasome knockouts, and identify ribosome mutants in the process reveal a major lethality and infertility hurdle that such designs must overcome to develop robust drives in the future. Although we do not achieve drive, this work provides a new tool for constructing future evolution-robust drive systems and reveals critical challenges that must be overcome for development of future gene drives designed to target haplolethal loci in anophelines and, potentially, other metazoans.
Gene drivesAnophelesCRISPRGene drive
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ACS synthetic biology · 2025-Nov-21 · PMID 41202215
Tianqi Wang, Daniel J Brogan, Natalie M Zawalick, Fangying Chen, Yeran Mkrtchian, Alexis C Komor, Omar S Akbari
Advancements in synthetic biology have enabled the development of precision gene expression technologies for comprehensive investigations of biological and biochemical networks. Here, we describe the development of a refined and innovative tool, CRISPR-Cas Transgenic Repressible eLement (CTRL), which utilizes the direct repeat processing activity of the recently discovered CRISPR-Cas7-11 effector to site-specifically target synthetic mRNA molecules. We demonstrate that CTRL exhibits high efficiency, tunable regulation of expression, and gene-specific repression of mRNA and protein expression. We engineered multiple permutations of the Cas7-11 effector that differ in their ability to reduce gene expression, suggesting flexibility for the application of choice. CTRL is a novel variation on gene repression technology that exhibits broad applicability across multiple model systems.
Synthetic biologyCRISPRTransgenesis
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Journal of visualized experiments : JoVE · 2025-Mar-21 · PMID 40193303
Iliano V Coutinho-Abreu, Fangying Chen, Hsing-Han Li, Noah H Rose, Omar S Akbari
The emergence of the clustered, regularly interspersed, short palindromic repeats (CRISPR)-Cas9 technology has revolutionized the genetic engineering field and opened the doors for precise genome editing in multiple species, including non-model organisms. In the mosquito Aedes aegypti, loss-of-function mutations and DNA insertions have been accomplished with this technology. Here, we describe a detailed protocol for genome editing through embryonic microinjection in the mosquito A. aegypti using the CRISPR-Cas9 technology, focusing on both the generation of gene knockout and knockin lines. In this protocol, quartz needles are filled with a mixture of guide RNA, recombinant Cas9, and a plasmid containing a DNA cassette encoding a gene for a fluorescent marker, if gene knockin is desired. Embryos at the preblastoderm stage are lined up onto a strip of double-sided sticky tape placed onto a coverslip, which is subsequently mounted onto a glass slide. With the help of a microinjector, the needles are inserted gently into the posterior end of the embryos and a small volume of the CRISPR mixture is dispensed. When the embryos are hatched, the larvae are checked under the fluorescent scope, and the pupae are sex-sorted and separated in different cages. Once the adults emerge, these are reciprocally crossed with wild-type individuals, blood-fed, and placed for egg laying. Once these eggs are hatched, the fluorescent larvae collected represent individuals with stable insertion of the DNA cassette into their genome. These larvae are then grown to the adult stage, outcrossed to wild-type individuals, and then further assessed through molecular techniques to confirm that the exact sequence of the DNA cassette is present at the desired site of the mosquito genome. Homozygous lines can also be obtained by following the provided pipeline of crossing schema and molecular screening of the mutations.
Synthetic biologyAedes aegyptiMosquitoesCRISPR
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G3 (Bethesda, Md.) · 2025-Mar-18 · PMID 39853276
Shruti Shankar, Diego Giraldo, Genevieve M Tauxe, Emma D Spikol, Ming Li, Omar S Akbari, Margot P Wohl, Conor J McMeniman
The mosquito Aedes aegypti is an emerging model insect for invertebrate neurobiology. We detail the application of a dual transgenesis marker system that reports the nature of transgene integration with circular donor template for CRISPR-Cas9-mediated homology-directed repair at target mosquito chemoreceptor genes. Employing this approach, we demonstrate the establishment of cell-type-specific T2A-QF2 driver lines for the A. aegypti olfactory co-receptor genes Ir8a and orco via canonical homology-directed repair and the CO2 receptor complex gene Gr1 via noncanonical homology-directed repair involving duplication of the intended T2A-QF2 integration cassette separated by intervening donor plasmid sequence. Using Gr1+ olfactory sensory neurons as an example, we show that introgression of such T2A-QF2 driver and QUAS responder transgenes into a yellow cuticular pigmentation mutant strain facilitates transcuticular calcium imaging of CO2-evoked neural activity on the maxillary palps with enhanced sensitivity relative to wild-type mosquitoes enveloped by dark melanized cuticle. We further apply Cre-loxP excision to derive marker-free T2A-QF2 in-frame fusions to clearly map axonal projection patterns from olfactory sensory neurons expressing these 3 chemoreceptors into the A. aegypti antennal lobe devoid of background interference from 3xP3-based fluorescent transgenesis markers. The marker-free Gr1 T2A-QF2 driver facilitates clear recording of CO2-evoked responses in this central brain region using the genetically encoded calcium indicators GCaMP6s and CaMPARI2. Systematic application of these optimized methods to different chemoreceptors stands to enable mapping A. aegypti olfactory circuits at peripheral and central levels of olfactory coding at high resolution.
Aedes aegyptiMosquitoesCRISPRTransgenesis
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Parasites & vectors · 2025-Jul-03 · PMID 40611351
Andrea L Smidler, Omar S Akbari
Malaria is one of the deadliest diseases on the planet, killing approximately 600,000 people annually, and is transmitted by the bite of an anopheline mosquito. Anophelines, and the diseases they transmit, have changed the course of history and the fate of nations, and their successful control promises to end the transmission of malaria. With the advent of clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) technologies, the study and control of these deadly pests have been revolutionized. As the release of genetically modified anophelines is being considered, here we outline the advances in CRISPR/Cas9 technologies and how they have revolutionized the study of anopheline basic biology and the development of innovative vector control strategies. We outline the major findings of CRISPR-based basic biological research into traits relevant for vector control including, but not limited to, olfaction, chemosensation, neurobiology, and reproduction. Further, we summarize the advancements in CRISPR-based innovative vector control strategies, such as the precision-guided sterile insect technique (pgSIT), inherited female elimination by genetically encoded nucleases to interrupt alleles (IFEGENIA), X-shredder, Y-linked editors, and gene drives. All in all, this review summarizes the basic biological and vector control research undertaken using CRISPR since its advent approximately a decade ago.
Sterile insect techniquesGene drivesMosquito neurobiologyVector controlMosquitoesCRISPR
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Cell · 2025-Dec-11 · PMID 41172998
Olivia V Goldman, Alexandra E DeFoe, Yanyan Qi, Yaoyu Jiao, Shih-Che Weng, Brittney Wick, Leah Houri-Zeevi, Priyanka Lakhiani, Takeshi Morita, Jacopo Razzauti, Adriana Rosas-Villegas, Yael N Tsitohay, Madison M Walker, Ben R Hopkins, Aedes aegypti Mosquito Cell Atlas Consortium, Maximilian Haeussler, Omar S Akbari, Laura B Duvall, Helen White-Cooper, Trevor R Sorrells, Roshan Sharma, Hongjie Li, Leslie B Vosshall, Nadav Shai
The female Aedes aegypti mosquito's remarkable ability to hunt humans and transmit pathogens relies on her unique biology. Here, we present the Aedes aegypti Mosquito Cell Atlas, a comprehensive single-nucleus RNA sequencing dataset of more than 367,000 nuclei from 19 dissected tissues of adult female and male Aedes aegypti, providing cellular-level resolution of mosquito biology. We identify novel cell types and expand our understanding of sensory neuron organization of chemoreceptors across all sensory tissues. Our analysis uncovers male-specific cells and sexually dimorphic gene expression in the antenna and brain. In female mosquitoes, we find that glial cells, rather than neurons, undergo the most extensive transcriptional changes in the brain following blood feeding. Our findings provide insights into the cellular basis of mosquito behavior and sexual dimorphism. The Aedes aegypti Mosquito Cell Atlas resource enables systematic investigation of cell-type-specific expression across all mosquito tissues.
Aedes aegyptiMosquitoes
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Parasites & vectors · 2025-Dec-05 · PMID 41351054
Sangwoo Seok, Miles T McCollum, Christopher M Jacobsen, Omar S Akbari, Derrick K Mathias, Yoosook Lee
Aedes albopictus (Skuse, 1894) and Aedes aegypti (Linnaeus, 1762) (Diptera: Culicidae) are invasive species in the Hawaiian Islands as well as other islands of the Pacific and serve as the primary vectors of arboviruses such as dengue virus. Despite its significance to public health, data on their insecticide resistance remains limited. Knowledge of the level of insecticide resistance is critical in developing effective mosquito control strategies, especially when an arboviral disease outbreak occurs.
This study investigated the phenotypic and genotypic resistance of Hawaiian Ae. albopictus and Ae. aegypti to permethrin, one of the commonly used pyrethroids. Genomic sequences of 93 Ae. albopictus samples from four Hawaiian Islands (Kaua'i, O'ahu, Maui, and the Island of Hawai'i) were screened for non-synonymous mutations in the voltage-gated sodium channel (VGSC) gene (chromosome 3: 345,142,499-345,663,668). Phenotypic resistance to permethrin was assessed using a modified Centers for Disease Control and Prevention (CDC) bottle bioassay on Ae. albopictus and Ae. aegypti collected from two socio-environmentally distinct sites on the Island of Hawai'i.
Among 4101 single-nucleotide polymorphisms (SNPs) identified in the VGSC region of Ae. albopictus genomes from four Hawaiian Islands, 44 were classified as synonymous in the coding DNA sequence (CDS) region. No non-synonymous mutations were found in the CDS region, suggesting an absence of genotypic resistance to pyrethroids in these populations. In phenotypic assays, all individuals from the two Ae. aegypti populations and the Ae. albopictus population from Miloli'i were completely knocked down within 10 min of permethrin exposure, indicating that both species remain phenotypically susceptible to permethrin. However, knockdown rates of less than 97% observed in the Ae. albopictus population from Kawaihae suggest early signs of the development of phenotypic resistance.
This study is the first to report the phenotypic insecticide resistance profile of Hawaiian Aedes mosquitoes. Hawaiian populations of Ae. albopictus and Ae. aegypti remain susceptible or are in the stage of developing resistance to pyrethroids, as demonstrated by the absence of VGSC mutations and high knockdown rates in permethrin bioassays. While no genotypic and phenotypic resistance was detected in these two Aedes species, monitoring for resistance in other mosquito species and through alternative mechanisms is needed.
Antiviral engineeringAedes aegyptiAedes albopictusMosquitoes
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PLoS neglected tropical diseases · 2025-Aug-11 · PMID 40788925
Sangwoo Seok, Adam E Vorsino, Travis C Collier, Limb K Hapairai, Christopher M Jacobsen, Jeomhee M Hasty, Ana L Romero-Weaver, Eva A Buckner, Dennis A LaPointe, Mark K H Leong, Leo Braack, Christine A Tabuloc, Joanna C Chiu, Robyn Raban, Omar S Akbari, Yoosook Lee
Remote Pacific islands (RPI) are characterized by ecological isolation, diverse endemic species, and vulnerability to invasive organisms due to globalization-driven connectivity. Among these species, Aedes albopictus, a highly invasive vector of flaviviruses, has spread extensively across the RPI via human-mediated dispersal, posing significant health and economic burdens. While the population structure and the degree of gene flow between mosquito populations can inform the dispersal pathways critical for disease vector management, the population genetics of Ae. albopictus in Northern RPI remains understudied. The present work investigated the population structure and connectivity of Ae. albopictus populations from Guam, Hawaiian Islands, and the Republic of the Marshall Islands (RMI) to inform disease and vector-based biosecurity risks and develop targeted management strategies. This is the first assessment to develop and analyze whole genome sequences of Ae. albopictus for RPI, enabling more accurate estimates of differentiation, admixture, and ancestry. We found distinct genetic clustering between regions, distinct ancestry of populations across RPI, and potential invasions that originated from Hawaii and spread into the RMI, and invasions from North America that spread to Guam. These findings can inform biosecurity protocols to limit the invasion of Ae. albopictus and their associated diseases within Hawaii and around the Pacific. Given the significant degree of genetic differentiation, we found between islets, islands, and regions, the genome data from this study can be used to enable the development of locally confined geographically isolated gene drives. These drives may be used to prevent and control outbreaks of dengue, chikungunya, and Zika, diseases that have had devastating consequences in these remote island communities.
Gene drivesAntiviral engineeringAedes albopictusMosquitoesGene drive
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G3 (Bethesda, Md.) · 2025-Aug-06 · PMID 40408551
Rhodri T M Edwards, Igor Antoshechkin, Eddie Hill, Michael W Perry, Pia U Olafson, Perot Saelao, Kimberly H Lohmeyer, Omar S Akbari
The winter tick, Dermacentor albipictus, is a significant North American ectoparasite, posing health risks to ruminants and occasionally humans. Despite its ecological importance, limited genomic resources exist for this species. This study provides the first comprehensive transcriptomic analysis of the recently published winter tick genome, focusing on tissues isolated from early-stage embryos, sexed adults, dissected ovaries, and dissected male reproductive systems. We identified the most abundant gene ontologies and analyzed differential gene expression. Differential gene expression revealed significant sex-biased expression patterns, and functional annotations identified candidate genes involved in sex determination. Notably, we identified the first documented case of sex-specific splicing of a doublesex-like gene in chelicerates, a mechanism previously thought to be absent in this clade. These transcriptome data serve as a critical resource for understanding the biology of D. albipictus and will facilitate the development of novel genetic technologies aimed at population suppression and reduction of tick-borne illnesses.
Sex determinationVector control
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BMC biology · 2025-Apr-12 · PMID 40221789
Serafima Davydova, Junru Liu, Yiran Liu, Kavya Prince, Jonathan Mann, Nikolay P Kandul, W Evan Braswell, Jackson Champer, Omar S Akbari, Angela Meccariello
Genetic biocontrol systems have broad applications in population control of insects implicated in both disease spread and food security. Ceratitis capitata (the Mediterranean fruit fly), a major agricultural pest with a global distribution, is one of the appealing targets for such genetic control.
In this study, we establish and characterise a novel split-CRISPR/Cas9 system we term Sex Conversion Induced by CRISPR (SCIC) in C. capitata. Using the white eye gene for toolkit selection we achieved up to 100% CRISPR/Cas9 efficiency, displaying the feasibility of C. capitata split-CRISPR/Cas9 systems using constitutive promoters. We then induce sex conversion by targeting the transformer gene in a SCIC approach aimed for SIT-mediated releases upon radiation-based sterilisation. Knock-out of transformer induced partial to full female-to-male sex conversion, with the remaining individuals all being intersex and sterile. SCIC population modelling shows a strong potential to outcompete traditional SIT, allowing for faster population elimination with fewer released sterile males.
Overall, we construct an appropriate CRISPR/Cas9 toolkit for the use in C capitata. Our results build the foundation for further genetic pest control methods in the species and related tephritid agricultural pests.
Sterile insect techniquesVector controlCRISPRSIT
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Nucleic acids research · 2025-Apr-10 · PMID 40226913
Calvin P Lin, Harry Li, Daniel J Brogan, Tianqi Wang, Omar S Akbari, Elizabeth A Komives
Type III-E CRISPR-Cas effectors, referred to as Cas7-11 or giant Repeat-Associated Mysterious Protein, are single proteins that cleave target RNAs (tgRNAs) without nonspecific collateral cleavage, opening new possibilities for RNA editing. Here, biochemical assays combined with amide hydrogen-deuterium exchange mass spectrometry (HDX-MS) experiments reveal the dynamics of apo Cas7-11. The HDX-MS results suggest a mechanism by which CRISPR RNA (crRNA) stabilizes the folded state of the protein and subsequent tgRNA binding remodels it to the active form. HDX-MS shows that the four Cas7 RNA recognition motif (RRM) folds are well-folded, but insertion sequences, including disordered catalytic loops and β-hairpins of the Cas7.2/Cas7.3 active sites, fold upon binding crRNA leading to stronger interactions at domain-domain interfaces, and folding of the Cas7.1 processing site. TgRNA binding causes conformational changes around the catalytic loops of Cas7.2 and Cas7.3. We show that Cas7-11 cannot independently process the CRISPR array and that binding of partially processed crRNA induces multiple states in Cas7-11 and reduces tgRNA cleavage. The insertion domain interacts most stably with mature crRNA. Finally, we show a crRNA-induced conformational change in one of the tetratricopeptide repeat fused with Cas/HEF1-associated signal transducer (TPR-CHAT) binding sites providing an explanation for why crRNA binding facilitates TPR-CHAT binding.
CRISPR
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PLOS global public health · 2025-07-18 · PMID 40680098
William A C Gendron, Robyn Raban, Agastya Mondal, Héctor M Sánchez C, Andrea L Smidler, David Zilberman, Patrick G C Ilboudo, Umberto D'Alessandro, John M Marshall, Omar S Akbari
Mosquito control has successfully reduced the burden of malaria globally, but current vector control technologies cannot achieve malaria elimination. Precision guided sterile insect technique (pgSIT) is one of the most promising interventions being developed for malaria elimination. Mass release of genetically sterile males can act as a chemical-free species-specific insecticide. Before translating pgSIT from the bench to the field, however, it is essential to understand the potential costs and capabilities of this technology in a malaria-endemic region to determine if further investment into research and development of this technology is worthwhile. Therefore, we estimated the health outcomes and costs of a pgSIT program working jointly with current interventions to control the Anopheles gambiae malaria vector in the Upper River region of The Gambia. The pgSIT intervention in this region is predicted to prevent approximately 230 deaths and about 48,000 sick days per year. We have provided a range of costs that include risks associated with research and development, the facility, mass rearing efficiency, and distribution. This intervention should save disability-adjusted life years (DALY) at 11-94 USD per year and will prevent cases at 10-86 USD per infection. These estimates predict that pgSIT will cost 0.36-3.03 USD per person in the treated region annually. The cost per DALY shows life-saving at a cost comparable to current interventions in the Upper River region.
Sterile insect techniquesVector controlAnophelesMosquitoespgSITSIT
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Communications biology · 2024-Oct-04 · PMID 39363033
Zihan Wei, Yaqi Wang, Kangwu Zheng, Zhiping Wang, Ronghua Liu, Pengcheng Wang, Yuting Li, Ping Gao, Omar S Akbari, Xueqing Yang
Genetic biocontrol technologies present promising and eco-friendly strategies for the management of pest and insect-transmitted diseases. Although considerable advancements achieve in gene drive applications targeting mosquitoes, endeavors to combat agricultural pests have been somewhat restricted. Here, we identify that the testis-specific serine/threonine kinases (TSSKs) family is uniquely expressed in the testes of Cydia pomonella, a prominent global invasive species. We further generated male moths with disrupted the expression of TSSKs and those with TSSKs disrupted using RNA interference and CRISPR/Cas9 genetic editing techniques, resulting in significant disruptions in spermiogenesis, decreased sperm motility, and hindered development of eggs. Further explorations into the underlying post-transcriptional regulatory mechanisms reveales the involvement of lnc117962 as a competing endogenous RNA (ceRNA) for miR-3960, thereby regulating TSSKs. Notably, orchard trials demonstrates that the release of male strains can effectively suppress population growth. Our findings indicate that targeting TSSKs could serve as a feasible avenue for managing C. pomonella populations, offering significant insights and potential strategies for controlling invasive pests through genetic sterile insect technique (gSIT) technology.
Sterile insect techniquesGene drivesMosquitoesCRISPRGene driveSIT
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Infectious diseases of poverty · 2024-Nov-11 · PMID 39523387
Shih-Che Weng, Fangying Chen, Ming Li, Sammy Lee, Connor Gerry, Dylan Can Turksoy, Omar S Akbari
Genetic biocontrol interventions targeting mosquito-borne diseases require the release of male mosquitoes exclusively, as only females consume blood and transmit pathogens. Releasing only males eliminates the risk of increasing mosquito bites and spreading pathogens while enabling effective population control. The aim of this study is to develop robust sex-sorting methods for early larval stages in mosquitoes, enabling scalable male-only releases for genetic biocontrol interventions.
To address the challenge of sex-sorting in the Asian malaria vector Anopheles stephensi, we engineer Sexing Element Produced by Alternative RNA-splicing of a Transgenic Observable Reporter (SEPARATOR). This dominant fluorescent-based method, previously proven effective in Aedes aegypti, exploits sex-specific alternative splicing of a reporter to ensure exclusive male-specific expression early in development. The sex-specific alternative RNA splicing of the doublesex gene was selected as a target for engineering SEPARATOR due to its evolutionary conservation in insects. To expand SEPARATOR's applicability for genetic sexing, we assessed the cross-species sex-specific RNA splicing activity of the An. gambiae doublesex (AngDsx) splicing module in An. stephensi. Male-specific enhanced green fluorescent protein (EGFP) expression was verified throughout the mosquito life cycle using a fluorescent stereomicroscope.
Our results confirm that SEPARATOR regulates male-specific EGFP expression in An. stephensi and enables reliable positive male selection from the first instar larval stages. Molecular analysis demonstrates that male-specific EGFP expression is dependent on doublesex sex-specific splicing events. Additionally, the splicing module from An. gambiae operates effectively in An. stephensi, demonstrating evolutionary conservation in sex-specific splicing events between these species.
SEPARATOR's independence from sex-chromosome linkage provides resistance to breakage that could be mediated by meiotic recombination and chromosomal rearrangements, making it highly suitable for mass male releases. By enabling precise male selection from the first instar larval stages, SEPARATOR represents a significant advancement that will aid in the genetic biocontrol for Anopheles mosquitoes.
Sex determinationVector controlSynthetic biologyAedes aegyptiAnophelesMosquitoes
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Cold Spring Harbor protocols · 2024-Mar-01 · PMID 38233222
Ruichen Sun, Robyn Raban, Omar S Akbari
No abstract is available from PubMed.
Aedes aegyptiCRISPR
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Cold Spring Harbor protocols · 2024-Mar-01 · PMID 38233223
Ruichen Sun, Robyn Raban, Omar S Akbari
No abstract is available from PubMed.
Synthetic biologyAedes aegyptiCRISPRTransgenesis
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PNAS nexus · 2024-Jul-30 · PMID 39081786
Feng Liu, Iliano V Coutinho-Abreu, Robyn Raban, Tam Thuy Dan Nguyen, Alejandra R Dimas, Joseph A Merriman, Omar S Akbari
The skin microbiome plays a pivotal role in the production of attractive cues detected by mosquitoes. Here, we leveraged recent advances in genetic engineering to significantly reduce the production of L-(+)-lactic acid as a strategy to reduce mosquito attraction to the highly prominent skin commensals Staphylococcus epidermidis and Corynebacterium amycolatum. Engraftment of these engineered bacteria onto the skin of mice reduced mosquito attraction and feeding for up to 11 uninterrupted days, which is considerably longer than the several hours of protection conferred by the leading chemical repellent N,N-diethyl-meta-toluamide. Taken together, our findings demonstrate engineering the skin microbiome to reduce attractive volatiles represents an innovative untapped strategy to reduce vector attraction, preventing bites, and pathogen transmission. These findings set the stage for new classes of long-lasting microbiome-based repellent products.
Synthetic biologyMosquitoes
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Proceedings of the National Academy of Sciences of the United States of America · 2024-Jul-02 · PMID 38917000
Reema A Apte, Andrea L Smidler, James J Pai, Martha L Chow, Sanle Chen, Agastya Mondal, Héctor M Sánchez C, Igor Antoshechkin, John M Marshall, Omar S Akbari
Controlling the principal African malaria vector, the mosquito Anopheles gambiae , is considered essential to curtail malaria transmission. However, existing vector control technologies rely on insecticides, which are becoming increasingly ineffective. Sterile insect technique (SIT) is a powerful suppression approach that has successfully eradicated a number of insect pests, yet the A. gambiae toolkit lacks the requisite technologies for its implementation. SIT relies on iterative mass releases of nonbiting, nondriving, sterile males which seek out and mate with monandrous wild females. Once mated, females are permanently sterilized due to mating-induced refractoriness, which results in population suppression of the subsequent generation. However, sterilization by traditional methods renders males unfit, making the creation of precise genetic sterilization methods imperative. Here, we introduce a vector control technology termed precision-guided sterile insect technique (pgSIT), in A. gambiae for inducible, programmed male sterilization and female elimination for wide-scale use in SIT campaigns. Using a binary CRISPR strategy, we cross separate engineered Cas9 and gRNA strains to disrupt male-fertility and female-essential genes, yielding >99.5% male sterility and >99.9% female lethality in hybrid progeny. We demonstrate that these genetically sterilized males have good longevity, are able to induce sustained population suppression in cage trials, and are predicted to eliminate wild A. gambiae populations using mathematical models, making them ideal candidates for release. This work provides a valuable addition to the malaria genetic biocontrol toolkit, enabling scalable SIT-like confinable, species-specific, and safe suppression in the species.
Sterile insect techniquesVector controlAnophelesMosquitoesCRISPRpgSIT
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eLife · 2024-Jan-30 · PMID 38289340
Ming Li, Nikolay P Kandul, Ruichen Sun, Ting Yang, Elena D Benetta, Daniel J Brogan, Igor Antoshechkin, Héctor M Sánchez C, Yinpeng Zhan, Nicolas A DeBeaubien, YuMin M Loh, Matthew P Su, Craig Montell, John M Marshall, Omar S Akbari
Each year, hundreds of millions of people are infected with arboviruses such as dengue, yellow fever, chikungunya, and Zika, which are all primarily spread by the notorious mosquito Aedes aegypti . Traditional control measures have proven insufficient, necessitating innovations. In response, here we generate a next-generation CRISPR-based precision-guided sterile insect technique (pgSIT) for Ae. aegypti that disrupts genes essential for sex determination and fertility, producing predominantly sterile males that can be deployed at any life stage. Using mathematical models and empirical testing, we demonstrate that released pgSIT males can effectively compete with, suppress, and eliminate caged mosquito populations. This versatile species-specific platform has the potential for field deployment to effectively control wild populations of disease vectors.
Sterile insect techniquesSex determinationAntiviral engineeringAedes aegyptiMosquitoesCRISPR
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Trends in parasitology · 2024-Jan-24 · PMID 38000957
Shih-Che Weng, Reem A Masri, Omar S Akbari
Mosquito-borne illnesses represent a significant global health peril, resulting in approximately one million fatalities annually. West Nile, dengue, Zika, and malaria are continuously expanding their global reach, driven by factors that escalate mosquito populations and pathogen transmission. Innovative control measures are imperative to combat these catastrophic ailments. Conventional approaches, such as eliminating breeding sites and using insecticides, have been helpful, but they face challenges such as insecticide resistance and environmental harm. Given the mounting severity of mosquito-borne diseases, there is promise in exploring innovative approaches using synthetic biology to bolster mosquitoes' resistance to pathogens, or even eliminate the mosquito vectors, as a means of control. This review outlines current strategies, future goals, and the importance of gene editing for global health defenses against mosquito-borne diseases.
Antiviral engineeringSynthetic biologyMosquitoes
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Scientific reports · 2024-Jan-18 · PMID 38238397
Iliano V Coutinho-Abreu, Omid Jamshidi, Robyn Raban, Katayoon Atabakhsh, Joseph A Merriman, Omar S Akbari
The resident human skin microbiome is responsible for the production of most of the human scents that are attractive to mosquitoes. Hence, engineering the human skin microbiome to synthesize less of mosquito attractants or produce repellents could potentially reduce bites and prevent the transmission of deadly mosquito-borne pathogens. In order to further characterize the human skin volatilome, we quantified the major volatiles of 39 strains of skin commensals (Staphylococci and Corynebacterium). Importantly, to validate the behavioral activity of these volatiles, we first assessed landing behavior triggered by human skin volatiles. We demonstrated that landing behavior is gated by the presence of carbon dioxide and L-(+)-lactic acid. This is similar to the combinatorial coding triggering mosquito short range attraction. Repellency behavior to selected skin volatiles and terpenes was tested in the presence of carbon dioxide and L-(+)-lactic acid. In a 2-choice landing behavior context, the skin volatiles 2- and 3-methyl butyric acids reduced mosquito landing by 62.0-81.6% and 87.1-99.6%, respectively. Similarly, the terpene geraniol was capable of reducing mosquito landing behavior by 74.9%. We also tested the potential repellency effects of terpenes in mosquitoes at short-range using a 4-port olfactometer. In these assays, geraniol reduced mosquito attraction (69-78%) to a mixture of key human kairomones carbon dioxide, L-(+)-lactic acid, and ammonia. These findings demonstrate that carbon dioxide and L-(+)-lactic acid change the valence of other skin volatiles towards mosquito landing behavior. Moreover, this study offers candidate odorants to be targeted in a novel strategy to reduce attractants or produce repellents by the human skin microbiota that may curtail mosquito bites, and subsequent mosquito-borne disease.
Mosquito neurobiologyMosquitoes
View paper record →laboratoryAbstract onlycorrected
Genome research · 2024-Feb-07 · PMID 38326031
Qicheng Ma, Satyam P Srivastav, Stephanie Gamez, Gargi Dayama, Fabiana Feitosa-Suntheimer, Edward I Patterson, Rebecca M Johnson, Erik M Matson, Alexander S Gold, Douglas E Brackney, John H Connor, Tonya M Colpitts, Grant L Hughes, Jason L Rasgon, Tony Nolan, Omar S Akbari, Nelson C Lau
No abstract is available from PubMed.
Antiviral engineeringMosquitoes
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GEN biotechnology · 2024-Feb-01 · PMID 38415050
Junru Liu, Danny Rayes, Omar S Akbari
Recent advances in insect genetic engineering offer alternative genetic biocontrol solutions to control populations of pests and disease vectors. While success has been achieved, sex-sorting remains problematic for scaling many genetic biocontrol interventions. Here, we describe the development of a genetically stable sex-sorting technique for female and male selection with a proof of concept in Drosophila melanogaster termed SEPARATOR (Sexing Element Produced by Alternative RNA-splicing of A Transgenic Observable Reporter). This elegant approach utilizes dominantly expressed fluorescent proteins and differentially spliced introns to ensure sex-specific expression. The system has the potential for adaptability to various insect species and application for high-throughput insect sex-sorting.
Synthetic biologyDrosophilaTransgenesis
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Insect molecular biology · 2024-Aug-07 · PMID 38450861
Nadya Urakova, Renuka E Joseph, Allyn Huntsinger, Vanessa M Macias, Matthew J Jones, Leah T Sigle, Ming Li, Omar S Akbari, Zhiyong Xi, Konstantinos Lymperopoulos, Richard T Sayre, Elizabeth A McGraw, Jason L Rasgon
Multiple Wolbachia strains can block pathogen infection, replication and/or transmission in Aedes aegypti mosquitoes under both laboratory and field conditions. However, Wolbachia effects on pathogens can be highly variable across systems and the factors governing this variability are not well understood. It is increasingly clear that the mosquito host is not a passive player in which Wolbachia governs pathogen transmission phenotypes; rather, the genetics of the host can significantly modulate Wolbachia-mediated pathogen blocking. Specifically, previous work linked variation in Wolbachia pathogen blocking to polymorphisms in the mosquito alpha-mannosidase-2 (αMan2) gene. Here we use CRISPR-Cas9 mutagenesis to functionally test this association. We developed αMan2 knockouts and examined effects on both Wolbachia and virus levels, using dengue virus (DENV; Flaviviridae) and Mayaro virus (MAYV; Togaviridae). Wolbachia titres were significantly elevated in αMan2 knockout (KO) mosquitoes, but there were complex interactions with virus infection and replication. In Wolbachia-uninfected mosquitoes, the αMan2 KO mutation was associated with decreased DENV titres, but in a Wolbachia-infected background, the αMan2 KO mutation significantly increased virus titres. In contrast, the αMan2 KO mutation significantly increased MAYV replication in Wolbachia-uninfected mosquitoes and did not affect Wolbachia-mediated virus blocking. These results demonstrate that αMan2 modulates arbovirus infection in A. aegypti mosquitoes in a pathogen- and Wolbachia-specific manner, and that Wolbachia-mediated pathogen blocking is a complex phenotype dependent on the mosquito host genotype and the pathogen. These results have a significant impact for the design and use of Wolbachia-based strategies to control vector-borne pathogens.
Antiviral engineeringAedes aegyptiMosquitoesCRISPR
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Frontiers in physiology · 2024-03-28 · PMID 38606012
Angela Rouyar, Anandrao A Patil, Melissa Leon-Noreña, Ming Li, Iliano V Coutinho-Abreu, Omar S Akbari, Jeff A Riffell
The mosquito Aedes aegypti is an important vector of diseases including dengue, Zika, chikungunya, and yellow fever. Olfaction is a critical modality for mosquitoes enabling them to locate hosts, sources of nectar, and sites for oviposition. GABA is an essential neurotransmitter in olfactory processing in the insect brain, including the primary olfactory center, the antennal lobe. Previous work with Ae. aegypti has suggested that antennal lobe inhibition via GABA may be involved in the processing of odors. However, little is known about GABA receptor expression in the mosquito brain, or how they may be involved in odor attraction. In this context, generating mutants that target the mosquito's olfactory responses, and particularly the GABAergic system, is essential to achieve a better understanding of these diverse processes and olfactory coding in these disease vectors. Here we demonstrate the potential of a transgenic line using the QF2 transcription factor, GABA-B1 QF2-ECFP , as a new neurogenetic tool to investigate the neural basis of olfaction in Ae. aegypti. Our results show that the gene insertion has a moderate impact on mosquito fitness. Moreover, the line presented here was crossed with a QUAS reporter line expressing the green fluorescent protein and used to determine the location of the metabotropic GABA-B1 receptor expression. We find high receptor expression in the antennal lobes, especially the cell bodies surrounding the antennal lobes. In the mushroom bodies, receptor expression was high in the Kenyon cells, but had low expression in the mushroom body lobes. Behavioral experiments testing the fruit odor attractants showed that the mutants lost their behavioral attraction. Together, these results show that the GABA-B1 QF2-ECFP line provides a new tool to characterize GABAergic systems in the mosquito nervous system.
Antiviral engineeringMosquito neurobiologySynthetic biologyAedes aegyptiMosquitoesTransgenesis
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Cold Spring Harbor protocols · 2023-Sep-01 · PMID 36931732
Ruichen Sun, Robyn Raban, Omar S Akbari
Since the characterization of the CRISPR-Cas9 system in prokaryotes, it has become the prime choice in gene editing because of its exceptional flexibility, ease of use, high efficiency, and superior specificity. As a result, CRISPR-Cas9-mediated gene-editing technologies have enabled researchers not only to engineer transgenic animal strains with site-directed insertions more efficiently but also to generate desired mutants for previously intractable species. One such species is the invasive yellow fever mosquito, Aedes aegypti , which is notorious for its ability to transmit many blood-borne human pathogens. Methods for developing new transgenic strains of the yellow fever mosquito may aid in the effort to control its populations and provide significant benefits for the public. Here, we provide an overview of injection and noninjection methods for generating transgenic mosquitoes and also highlight important experimental design features.
Synthetic biologyAedes aegyptiMosquitoesCRISPRTransgenesis
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Cold Spring Harbor protocols · 2023-Sep-01 · PMID 36931733
Ruichen Sun, Robyn Raban, Omar S Akbari
Here, we provide a protocol for generating Aedes aegypti mutant strains via end-joining (EJ) or homology-directed repair (HDR) mechanisms using genetically encoded Cas9.
Synthetic biologyAedes aegyptiCRISPRTransgenesis
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PLoS genetics · 2023-Nov-27 · PMID 38011259
Shih-Che Weng, Igor Antoshechkin, Eric Marois, Omar S Akbari
Only female mosquitoes consume blood giving them the opportunity to transmit deadly human pathogens. Therefore, it is critical to remove females before conducting releases for genetic biocontrol interventions. Here we describe a robust sex-sorting approach termed SEPARATOR (Sexing Element Produced by Alternative RNA-splicing of A Transgenic Observable Reporter) that exploits sex-specific alternative splicing of an innocuous reporter to ensure exclusive dominant male-specific expression. Using SEPARATOR, we demonstrate reliable sex selection from early larval and pupal stages in Aedes aegypti, and use a Complex Object Parametric Analyzer and Sorter (COPAS) to demonstrate scalable high-throughput sex-selection of first instar larvae. Additionally, we use this approach to sequence the transcriptomes of early larval males and females and find several genes that are sex-specifically expressed. SEPARATOR can simplify mass production of males for release programs and is designed to be cross-species portable and should be instrumental for genetic biocontrol interventions.
Synthetic biologyAedes aegyptiMosquitoesTransgenesis
View paper record →reviewAbstract only
Annual review of genetics · 2023-Nov-27 · PMID 37722684
Robyn Raban, John M Marshall, Bruce A Hay, Omar S Akbari
Genetic biocontrol aims to suppress or modify populations of species to protect public health, agriculture, and biodiversity. Advancements in genome engineering technologies have fueled a surge in research in this field, with one gene editing technology, CRISPR, leading the charge. This review focuses on the current state of CRISPR technologies for genetic biocontrol of pests and highlights the progress and ongoing challenges of using these approaches.
CRISPR
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Scientific reports · 2023-Nov-14 · PMID 37964160
Serafima Davydova, Junru Liu, Nikolay P Kandul, W Evan Braswell, Omar S Akbari, Angela Meccariello
Tephritid fruit fly pests pose an increasing threat to the agricultural industry due to their global dispersion and a highly invasive nature. Here we showcase the feasibility of an early-detection SEPARATOR sex sorting approach through using the non-model Tephritid pest, Ceratitis capitata. This system relies on female-only fluorescent marker expression, accomplished through the use of a sex-specific intron of the highly-conserved transformer gene from C. capitata and Anastrepha ludens. The herein characterized strains have 100% desired phenotype outcomes, allowing accurate male-female separation during early development. Overall, we describe an antibiotic and temperature-independent sex-sorting system in C. capitata, which, moving forward, may be implemented in other non-model Tephritid pest species. This strategy can facilitate the establishment of genetic sexing systems with endogenous elements exclusively, which, on a wider scale, can improve pest population control strategies like sterile insect technique.
Sterile insect techniquesSex determinationVector controlSIT
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Nature communications · 2023-Mar-29 · PMID 36991021
Stephanie L James, David A O'Brochta, Filippo Randazzo, Omar S Akbari
We address a controversy over use of the term “gene drive” to include both natural and synthetic genetic elements that promote their own transmission within a population, arguing that this broad definition is both practical and has advantages for risk analysis.
Gene drivesGene drive
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Journal of medical entomology · 2023-Mar-06 · PMID 36656078
Katherine K Brisco, Christopher M Jacobsen, Sangwoo Seok, Xiaodi Wang, Yoosook Lee, Omar S Akbari, Anthony J Cornel
Aedes aegypti Linnaeus and Aedes albopictus Skuse are vectors of dengue virus and responsible for multiple autochthonous dengue outbreaks in Big Island, Hawai'i. Control of Ae. aegypti and Ae. albopictus has been achieved in In2Care trap trials, which motivated us to investigate this potential control approach in the Big Island. Our In2Care trial was performed in the coastal settlement of Miloli'i in the southwest of Big Island where both Ae. aegypti and Ae. albopictus are found. This trial starting in the second week of July and ending in the last week of October 2019 fell within the traditional wet season in Miloli'i. No significant reduction in egg or adult counts in our treatment areas following 12 wk of two In2Care trap placements per participating household were observed. In fact, an increase in numbers of adults during the trial reached levels that required the local mosquito abatement program to stop the In2Care trap trial and institute a thorough source reduction and treatment campaign. The source reduction campaign revealed a large variety and quantity of water sources competed with the oviposition cups we had placed, which likely lowered the chances of our oviposition cups being visited by pyriproxyfen-contaminated Aedes adults exiting the In2Care traps.
Antiviral engineeringAedes aegyptiAedes albopictusMosquitoes
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Communications biology · 2023-Jun-16 · PMID 37328568
Célia Lutrat, Myriam Burckbuchler, Roenick Proveti Olmo, Rémy Beugnon, Albin Fontaine, Omar S Akbari, Rafael Argilés-Herrero, Thierry Baldet, Jérémy Bouyer, Eric Marois
Chemical control of disease vectoring mosquitoes Aedes albopictus and Aedes aegypti is costly, unsustainable, and increasingly ineffective due to the spread of insecticide resistance. The Sterile Insect Technique is a valuable alternative but is limited by slow, error-prone, and wasteful sex-separation methods. Here, we present four Genetic Sexing Strains (two for each Aedes species) based on fluorescence markers linked to the m and M sex loci, allowing for the isolation of transgenic males. Furthermore, we demonstrate how combining these sexing strains enables the production of non-transgenic males. In a mass-rearing facility, 100,000 first instar male larvae could be sorted in under 1.5 h with an estimated 0.01-0.1% female contamination on a single machine. Cost-efficiency analyses revealed that using these strains could result in important savings while setting up and running a mass-rearing facility. Altogether, these Genetic Sexing Strains should enable a major upscaling in control programmes against these important vectors.
Sterile insect techniquesSex determinationSynthetic biologyAedes aegyptiAedes albopictusMosquitoes
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Science advances · 2023-Jul-07 · PMID 37406109
Andrea L Smidler, James J Pai, Reema A Apte, Héctor M Sánchez C, Rodrigo M Corder, Eileen Jeffrey Gutiérrez, Neha Thakre, Igor Antoshechkin, John M Marshall, Omar S Akbari
Malaria is among the world's deadliest diseases, predominantly affecting Sub-Saharan Africa and killing over half a million people annually. Controlling the principal vector, the mosquito Anopheles gambiae , as well as other anophelines, is among the most effective methods to control disease spread. Here, we develop a genetic population suppression system termed Ifegenia (inherited female elimination by genetically encoded nucleases to interrupt alleles) in this deadly vector. In this bicomponent CRISPR-based approach, we disrupt a female-essential gene, femaleless ( fle ), demonstrating complete genetic sexing via heritable daughter gynecide. Moreover, we demonstrate that Ifegenia males remain reproductively viable and can load both fle mutations and CRISPR machinery to induce fle mutations in subsequent generations, resulting in sustained population suppression. Through modeling, we demonstrate that iterative releases of nonbiting Ifegenia males can act as an effective, confinable, controllable, and safe population suppression and elimination system.
Sex determinationVector controlAnophelesMosquitoesCRISPR
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PLoS pathogens · 2023-Jan-19 · PMID 36656895
Michelle Bui, Elena Dalla Benetta, Yuemei Dong, Yunchong Zhao, Ting Yang, Ming Li, Igor A Antoshechkin, Anna Buchman, Vanessa Bottino-Rojas, Anthony A James, Michael W Perry, George Dimopoulos, Omar S Akbari
As a major insect vector of multiple arboviruses, Aedes aegypti poses a significant global health and economic burden. A number of genetic engineering tools have been exploited to understand its biology with the goal of reducing its impact. For example, current tools have focused on knocking-down RNA transcripts, inducing loss-of-function mutations, or expressing exogenous DNA. However, methods for transactivating endogenous genes have not been developed. To fill this void, here we developed a CRISPR activation (CRISPRa) system in Ae. aegypti to transactivate target gene expression. Gene expression is activated through pairing a catalytically-inactive ('dead') Cas9 (dCas9) with a highly-active tripartite activator, VP64-p65-Rta (VPR) and synthetic guide RNA (sgRNA) complementary to a user defined target-gene promoter region. As a proof of concept, we demonstrate that engineered Ae. aegypti mosquitoes harboring a binary CRISPRa system can be used to effectively overexpress two developmental genes, even-skipped (eve) and hedgehog (hh), resulting in observable morphological phenotypes. We also used this system to overexpress the positive transcriptional regulator of the Toll immune pathway known as AaRel1, which resulted in a significant suppression of dengue virus serotype 2 (DENV2) titers in the mosquito. This system provides a versatile tool for research pathways not previously possible in Ae. aegypti, such as programmed overexpression of endogenous genes, and may aid in gene characterization studies and the development of innovative vector control tools.
Antiviral engineeringVector controlSynthetic biologyAedes aegyptiMosquitoesCRISPR
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Nature biotechnology · 2023-Jan-16 · PMID 36522496
Riley I Taitingfong, Cynthia Triplett, Váleri N Vásquez, Ramya M Rajagopalan, Robyn Raban, Aaron Roberts, Gerard Terradas, Bridget Baumgartner, Claudia Emerson, Fred Gould, Fredros Okumu, Cynthia E Schairer, Hervé C Bossin, Leah Buchman, Karl J Campbell, Anna Clark, Jason Delborne, Kevin Esvelt, Joshua Fisher, Robert M Friedman, Gigi Gronvall, Nikos Gurfield, Elizabeth Heitman, Natalie Kofler, Todd Kuiken, Jennifer Kuzma, Pablo Manrique-Saide, John M Marshall, Michael Montague, Amy C Morrison, Chris C Opesen, Ryan Phelan, Antoinette Piaggio, Hector Quemada, Larisa Rudenko, Natéwindé Sawadogo, Robert Smith, Holly Tuten, Anika Ullah, Adam Vorsino, Nikolai Windbichler, Omar S Akbari, Kanya Long, James V Lavery, Sam Weiss Evans, Karen Tountas, Cinnamon S Bloss
No abstract is available from PubMed.
Gene drivesGene drive
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Trends in genetics : TIG · 2023-Feb-20 · PMID 36414481
Iliano V Coutinho-Abreu, Omar S Akbari
Gene-editing technologies have revolutionized the field of mosquito sensory biology. These technologies have been used to knock in reporter genes in-frame with neuronal genes and tag specific mosquito neurons to detect their activities using binary expression systems. Despite these advances, novel tools still need to be developed to elucidate the transmission of olfactory signals from the periphery to the brain. Here, we propose the development of a set of tools, including novel driver lines as well as sensors of neuromodulatory activities, which can advance our knowledge of how sensory input triggers behavioral outputs. This information can change our understanding of mosquito neurobiology and lead to the development of strategies for mosquito behavioral manipulation to reduce bites and disease transmission.
Mosquito neurobiologyMosquitoes
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Biochemistry · 2023-Dec-19 · PMID 35325535
Daniel J Brogan, Omar S Akbari
CRISPR diagnostics have recently emerged as powerful diagnostic tools for the rapid detection of infections. The ultimate goal is to develop these diagnostics for the point of care, where patients quickly receive and easily interpret results. Although they are in their infancy, the COVID-19 pandemic has accelerated innovation of CRISPR diagnostics and led to an explosion of improvements to these systems. Challenges that have impeded the implementation at the point of care have been addressed, and CRISPR diagnostics have been dramatically simplified. Here we outline recent developments and advancements in CRISPR diagnostics that have pushed these technologies to the point of care.
CRISPR
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The CRISPR journal · 2023-Dec-19 · PMID 38108518
Elena Dalla Benetta, Adam J López-Denman, Hsing-Han Li, Reem A Masri, Daniel J Brogan, Michelle Bui, Ting Yang, Ming Li, Michael Dunn, Melissa J Klein, Sarah Jackson, Kyle Catalan, Kim R Blasdell, Priscilla Tng, Igor Antoshechkin, Luke S Alphey, Prasad N Paradkar, Omar S Akbari
Escalating vector disease burdens pose significant global health risks, as such innovative tools for targeting mosquitoes are critical. CRISPR-Cas technologies have played a crucial role in developing powerful tools for genome manipulation in various eukaryotic organisms. Although considerable efforts have focused on utilizing class II type II CRISPR-Cas9 systems for DNA targeting, these modalities are unable to target RNA molecules, limiting their utility against RNA viruses. Recently, the Cas13 family has emerged as an efficient tool for RNA targeting; however, the application of this technique in mosquitoes, particularly Aedes aegypti , has yet to be fully realized. In this study, we engineered an antiviral strategy termed REAPER (vRNA Expression Activates Poisonous Effector Ribonuclease) that leverages the programmable RNA-targeting capabilities of CRISPR-Cas13 and its potent collateral activity. REAPER remains concealed within the mosquito until an infectious blood meal is uptaken. Upon target viral RNA infection, REAPER activates, triggering programmed destruction of its target arbovirus such as chikungunya. Consequently, Cas13-mediated RNA targeting significantly reduces viral replication and viral prevalence of infection, and its promiscuous collateral activity can even kill infected mosquitoes within a few days. This innovative REAPER technology adds to an arsenal of effective molecular genetic tools to combat mosquito virus transmission.
Antiviral engineeringAedes aegyptiMosquitoesCRISPR
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EBioMedicine · 2023-Aug-22 · PMID 37487418
Hsing-Han Li, Matthew P Su, Shih-Cheng Wu, Hsiao-Hui Tsou, Meng-Chun Chang, Yu-Chieh Cheng, Kuen-Nan Tsai, Hsin-Wei Wang, Guan-Hua Chen, Cheng-Kang Tang, Pei-Jung Chung, Wan-Ting Tsai, Li-Rung Huang, Yueh Andrew Yueh, Hsin-Wei Chen, Chao-Ying Pan, Omar S Akbari, Hsiao-Han Chang, Guann-Yi Yu, John M Marshall, Chun-Hong Chen
Dengue virus outbreaks are increasing in number and severity worldwide. Viral transmission is assumed to require a minimum time period of viral replication within the mosquito midgut. It is unknown if alternative transmission periods not requiring replication are possible.
We used a mouse model of dengue virus transmission to investigate the potential of mechanical transmission of dengue virus. We investigated minimal viral titres necessary for development of symptoms in bitten mice and used resulting parameters to inform a new model of dengue virus transmission within a susceptible population.
Naïve mice bitten by mosquitoes immediately after they took partial blood meals from dengue infected mice showed symptoms of dengue virus, followed by mortality. Incorporation of mechanical transmission into mathematical models of dengue virus transmission suggest that this supplemental transmission route could result in larger outbreaks which peak sooner.
The potential of dengue transmission routes independent of midgut viral replication has implications for vector control strategies that target mosquito lifespan and suggest the possibility of similar mechanical transmission routes in other disease-carrying mosquitoes.
This study was funded by grants from the National Health Research Institutes, Taiwan (04D2-MMMOST02), the Human Frontier Science Program (RGP0033/2021), the National Institutes of Health (1R01AI143698-01A1, R01AI151004 and DP2AI152071) and the Ministry of Science and Technology, Taiwan (MOST104-2321-B-400-016).
Antiviral engineeringVector controlAedes aegyptiMosquitoes
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G3 (Bethesda, Md.) · 2023-Apr-11 · PMID 36705519
Natalie Warsinger-Pepe, Carly Chang, Connor R Desroberts, Omar S Akbari
Heat-shock-inducible expression of genes through the use of heat-inducible promoters is commonly used in research despite leaky expression of downstream genes of interest without targeted induction (i.e. heat shock). The development of non-leaky inducible expression systems is of broad interest for both basic and applied studies, to precisely control gene expression. Here we characterize the use of Polycomb response elements and the inducible Heat-shock protein 70Bb promoter, previously described as a non-leaky inducible system, to regulate Cas9 endonuclease levels and function in Drosophila melanogaster after varying both heat-shock durations and rearing temperatures. We show that Polycomb response elements can significantly reduce expression of Cas9 under Heat-shock protein 70Bb promoter control using a range of conditions, corroborating previously published results. We further demonstrate that this low transcript level of heat-induced Cas9 is sufficient to induce mutant mosaic phenotypes. Incomplete suppression of an inducible Cas9 system by Polycomb response elements with no heat-shock suggests that further regulatory elements are required to precisely control Cas9 expression and abundance.
DrosophilaCRISPR
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Mitochondrial DNA. Part B, Resources · 2023-01-16 · PMID 36685646
Sangwoo Seok, Christopher M Jacobsen, Ana L Romero-Weaver, Xiaodi Wang, Valerie T Nguyen, Travis C Collier, Michael T Riles, Omar S Akbari, Yoosook Lee
We report the first complete mitogenome (Mt) sequence of Aedes japonicus japonicus (Diptera: Culicidae). The sequence was extracted from one adult from the Big Island of Hawai'i Island. The length of the Ae. japonicus japonicus Mt was 16,528bp with 78.1% AT content. Its sequence is most similar to the Mt sequence of Aedes koreicus with 90.81% sequence identity. This is the first full Mt sequence available for this species and provides important genetic resource for studying population genetics and dynamics of this important invasive mosquito species.
Mosquitoes
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Nature communications · 2022-Nov-21 · PMID 36414618
Sebald A N Verkuijl, Estela Gonzalez, Ming Li, Joshua X D Ang, Nikolay P Kandul, Michelle A E Anderson, Omar S Akbari, Michael B Bonsall, Luke Alphey
CRISPR/Cas gene drives can bias transgene inheritance through different mechanisms. Homing drives are designed to replace a wild-type allele with a copy of a drive element on the homologous chromosome. In Aedes aegypti, the sex-determining locus is closely linked to the white gene, which was previously used as a target for a homing drive element (w GDe ). Here, through an analysis using this linkage we show that in males inheritance bias of w GDe did not occur by homing, rather through increased propagation of the donor drive element. We test the same w GDe drive element with transgenes expressing Cas9 with germline regulatory elements sds3, bgcn, and nup50. We only find inheritance bias through homing, even with the identical nup50-Cas9 transgene. We propose that DNA repair outcomes may be more context dependent than anticipated and that other previously reported homing drives may, in fact, bias their inheritance through other mechanisms.
Gene drivesAedes aegyptiCRISPRGene drive
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Trends in parasitology · 2022-Mar-18 · PMID 34674963
Iliano V Coutinho-Abreu, Jeffrey A Riffell, Omar S Akbari
Female mosquitoes use chemical and physical cues, including vision, smell, heat, and humidity, to orient toward hosts. Body odors are produced by skin resident bacteria that convert metabolites secreted in sweat into odorants that confer the characteristic body scent. Mosquitoes detect these compounds using olfactory receptors in their antennal olfactory receptor neurons. Such information is further integrated with the senses of temperature and humidity, as well as vision, processed in the brain into a behavioral output, leading to host finding. Knowledge of human scent components unveils a variety of odorants that are attractive to mosquitoes, but also odor-triggering repellency. Finding ways to divert human-seeking behavior by female mosquitoes using odorants can possibly mitigate mosquito-borne pathogen transmission.
Mosquito neurobiologyMosquitoes
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Scientific reports · 2022-Jan-10 · PMID 35013523
Wei-Liang Liu, Chia-Wei Hsu, Shih-Peng Chan, Pei-Shi Yen, Matthew P Su, Jian-Chiuan Li, Hsing-Han Li, Lie Cheng, Cheng-Kang Tang, Shih-Hsun Ko, Huai-Kuang Tsai, Zing Tsung-Yeh Tsai, Omar S Akbari, Anna-Bella Failloux, Chun-Hong Chen
No abstract is available from PubMed.
Antiviral engineeringSynthetic biologyAedes aegyptiTransgenesis
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Insect molecular biology · 2022-Apr-25 · PMID 34919304
Sanja Djokic, Anna Bakhrat, Ming Li, Omar S Akbari, Uri Abdu
Insect epithelial cells contain unique cellular extensions such as bristles, hairs, and scales. In contrast to bristle and hair, which are not divergent in their shape, scale morphology shows high diversity. In our attempt to characterize the role of the insect-specific gene, Spindle-F (spn-F), in mosquito development, we revealed a scale-type specific requirement for the mosquito Aedes aegypti spn-F homologue. Using CRISPR-Cas9, we generated Ae-spn-F mutants and found that Ae-spn-F is an essential gene, but we were able to recover a few adult escapers. These escapers could not fly nor move, and died after 3 to 4 days. We found that in Ae-spn-F mutants, only the tip part of the bristle was affected with bulbous with misoriented ribs. We also show that in Ae-spn-F mutants, only in falcate scales, which are curved with a sharp or narrowly rounded apex, and not in other scale types, the tip region is strongly affected. Our analysis also revealed that in contrast to Drosophila spn-F, which show strong defects in both the actin and microtubule (MT) network in the bristle, the Ae-spn-F gene is required only for MT organization in scales and bristles. In summary, our results reveal that Ae-spn-F is required for shaping tapered epithelial cellular extension structures, namely, the bristle and falcate scales by affecting MT organization.
Aedes aegyptiDrosophilaMosquitoesCRISPR
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Methods in molecular biology (Clifton, N.J.) · 2022-7-7 · PMID 35796955
Ruichen Sun, Ming Li, Conor J McMeniman, Omar S Akbari
CRISPR-mediated genome engineering technologies have been adapted to a wide variety of organisms with high efficiency and specificity. The yellow fever mosquito, Aedes aegypti , is one such organism. It is also responsible for transmitting a wide variety of deadly viruses including Dengue, Zika, Yellow fever, and Chikungunya. The key to successful CRISPR-mediated gene editing applications is the delivery of both Cas9 ribonuclease and single-guide RNA (sgRNA ) to the nucleus of desired cells. Various methods have been developed for supplying the Cas9 endonuclease, sgRNA , and donor DNA to Ae. aegypti. In this chapter, we focus on methods of direct embryo delivery of editing components, presenting detailed step-by-step CRISPR/Cas9-based genome-editing protocols for inducing desired heritable edits in mosquitoes as well as insights into successful application of these protocols. We also highlight potential opportunities for customizing these protocols to manipulate the mosquito genome for innovative in vivo gene function studies.
Antiviral engineeringAedes aegyptiMosquitoesCRISPR
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Frontiers in bioengineering and biotechnology · 2022-03-10 · PMID 35360388
Cynthia E Schairer, Cynthia Triplett, Omar S Akbari, Cinnamon S Bloss
Scientists developing gene drive mosquitoes for vector control must understand how residents of affected areas regard both the problem of mosquito-borne disease and the potential solutions offered by gene drive. This study represents an experiment in public engagement at an early stage of technology development, intended to inform lab scientists about public attitudes toward their research and inspire consideration and conversation about the social ramifications of creating mosquitoes with gene drive. Online focus groups with California residents explored views on mosquito-borne disease risk, current mosquito control methods, and the proposed development and use of different classes of gene drives to control Ae. aegypti . Rather than a dogmatic rejection of genetic engineering or gene drive, many participants expressed pragmatic concerns with cost, control, the ability to narrowly target specific species, and the challenges of mistrust and institutional cooperation. Work like this can inform the alignment of community priorities and the professional priorities of scientists and vector control specialists.
Gene drivesVector controlSynthetic biologyAedes aegyptiMosquitoesGene drive
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Pathogens and global health · 2021-Sep-27 · PMID 34313556
Cynthia E Schairer, James Najera, Anthony A James, Omar S Akbari, Cinnamon S Bloss
In response to growing concerns regarding mosquito-borne diseases, scientists are developing novel systems of vector control. Early examples include Oxitec's OX513A genetically-engineered mosquito and MosquitoMate's Wolbachia -infected mosquito, and systems using 'gene-drive' are in development. Systems based on genetic engineering are controversial and institutions around the world are grappling with the question of who should have a say in how such technologies are field-tested and used. Based on media coverage and public records, we created comparative timelines of the efforts of Oxitec and MosquitoMate to navigate federal and local governance and bring their products to market in the United States. We analyze these timelines with particular attention to the role of public input in technology governance. These cases illustrate how governance of technology in the US is diverse, complex, and opaque. Further, the public response to proposed field trials of the Oxitec product highlights inconsistencies between public expectations for governance and actual practice. As gene-drive mosquito control products develop, both federal and local agencies will find their legitimacy tested without a better procedure for transparently integrating public input.
Gene drivesVector controlSynthetic biologyMosquitoesGene drive
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Nature communications · 2021-Sep-10 · PMID 34508072
Ming Li, Ting Yang, Michelle Bui, Stephanie Gamez, Tyler Wise, Nikolay P Kandul, Junru Liu, Lenissa Alcantara, Haena Lee, Jyotheeswara R Edula, Robyn Raban, Yinpeng Zhan, Yijin Wang, Nick DeBeaubien, Jieyan Chen, Héctor M Sánchez C, Jared B Bennett, Igor Antoshechkin, Craig Montell, John M Marshall, Omar S Akbari
The mosquito Aedes aegypti is the principal vector for arboviruses including dengue/yellow fever, chikungunya, and Zika virus, infecting hundreds of millions of people annually. Unfortunately, traditional control methodologies are insufficient, so innovative control methods are needed. To complement existing measures, here we develop a molecular genetic control system termed precision-guided sterile insect technique (pgSIT) in Aedes aegypti. PgSIT uses a simple CRISPR-based approach to generate flightless females and sterile males that are deployable at any life stage. Supported by mathematical models, we empirically demonstrate that released pgSIT males can compete, suppress, and even eliminate mosquito populations. This platform technology could be used in the field, and adapted to many vectors, for controlling wild populations to curtail disease in a safe, confinable, and reversible manner.
Sterile insect techniquesAntiviral engineeringAedes aegyptiMosquitoesCRISPRpgSIT
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PLoS pathogens · 2021-Oct-29 · PMID 34714893
Sophia C Parks, Susan Nguyen, Shyon Nasrolahi, Chaitra Bhat, Damian Juncaj, Dihong Lu, Raghavendran Ramaswamy, Harpal Dhillon, Hideji Fujiwara, Anna Buchman, Omar S Akbari, Naoki Yamanaka, Martin J Boulanger, Adler R Dillman
Parasitic nematodes cause significant morbidity and mortality globally. Excretory/secretory products (ESPs) such as fatty acid- and retinol- binding proteins (FARs) are hypothesized to suppress host immunity during nematode infection, yet little is known about their interactions with host tissues. Leveraging the insect parasitic nematode, Steinernema carpocapsae, we describe here the first in vivo study demonstrating that FARs modulate animal immunity, causing an increase in susceptibility to bacterial co-infection. Moreover, we show that FARs dampen key components of the fly immune response including the phenoloxidase cascade and antimicrobial peptide (AMP) production. Our data also reveal that FARs deplete lipid signaling precursors in vivo as well as bind to these fatty acids in vitro, suggesting that FARs elicit their immunomodulatory effects by altering the availability of lipid signaling molecules necessary for an efficient immune response. Collectively, these data support a complex role for FARs in immunosuppression in animals and provide detailed mechanistic insight into parasitism in phylum Nematoda.
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The CRISPR journal · 2021-Oct-18 · PMID 34661429
Nikolay P Kandul, Esther J Belikoff, Junru Liu, Anna Buchman, Fang Li, Akihiko Yamamoto, Ting Yang, Isaiah Shriner, Maxwell J Scott, Omar S Akbari
Originally from Asia, Drosophila suzukii Matsumura is a global pest of economically important soft-skinned fruits. Also commonly known as spotted wing drosophila, it is largely controlled through repeated applications of broad-spectrum insecticides by which resistance has been observed in the field. There is a pressing need for a better understanding of D. suzukii biology and for developing alternative environmentally friendly methods of control. The RNA-guided Cas9 nuclease has revolutionized functional genomics and is an integral component of several recently developed genetic strategies for population control of insects. Here, we describe genetically modified strains that encode three different terminators and four different promoters to express Cas9 robustly in both the soma and/or germline of D. suzukii . The Cas9 strains were rigorously evaluated through genetic crossing to transgenic strains that encode single-guide RNAs targeting the conserved X-linked yellow body and white eye genes. We find that several Cas9/gRNA strains display remarkably high editing capacity. Going forward, these tools will be instrumental for evaluating gene function in D. suzukii and may even provide tools useful for the development of new genetic strategies for control of this invasive species.
Vector controlSynthetic biologyDrosophilaCRISPRTransgenesis
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ACS sensors · 2021-Nov-26 · PMID 34714054
Daniel J Brogan, Duverney Chaverra-Rodriguez, Calvin P Lin, Andrea L Smidler, Ting Yang, Lenissa M Alcantara, Igor Antoshechkin, Junru Liu, Robyn R Raban, Pedro Belda-Ferre, Rob Knight, Elizabeth A Komives, Omar S Akbari
The development of an extensive toolkit for potential point-of-care diagnostics that is expeditiously adaptable to new emerging pathogens is of critical public health importance. Recently, a number of novel CRISPR-based diagnostics have been developed to detect SARS-CoV-2. Herein, we outline the development of an alternative CRISPR nucleic acid diagnostic utilizing a Cas13d ribonuclease derived from Ruminococcus flavefaciens XPD3002 (CasRx) to detect SARS-CoV-2, an approach we term SENSR (sensitive enzymatic nucleic acid sequence reporter) that can detect attomolar concentrations of SARS-CoV-2. We demonstrate 100% sensitivity in patient-derived samples by lateral flow and fluorescence readout with a detection limit of 45 copy/μL. This technology expands the available nucleic acid diagnostic toolkit, which can be adapted to combat future pandemics.
CRISPR
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Heredity · 2021-May-01 · PMID 33649572
Elena Dalla Benetta, Omar S Akbari, Patrick M Ferree
Insects naturally harbor a broad range of selfish agents that can manipulate their reproduction and development, often leading to host sex ratio distortion. Such effects directly benefit the spread of the selfish agents. These agents include two broad groups: bacterial symbionts and selfish chromosomes. Recent studies have made steady progress in uncovering the cellular targets of these agents and their effector genes. Here we highlight what is known about the targeted developmental processes, developmental timing, and effector genes expressed by several selfish agents. It is now becoming apparent that: (1) the genetic toolkits used by these agents to induce a given reproductive manipulation are simple, (2) these agents target sex-specific cellular processes very early in development, and (3) in some cases, similar processes are targeted. Knowledge of the molecular underpinnings of these systems will help to solve long-standing puzzles and provide new tools for controlling insect pests.
Sex determination
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Genome research · 2021-Mar-08 · PMID 33419731
Qicheng Ma, Satyam P Srivastav, Stephanie Gamez, Gargi Dayama, Fabiana Feitosa-Suntheimer, Edward I Patterson, Rebecca M Johnson, Erik M Matson, Alexander S Gold, Douglas E Brackney, John H Connor, Tonya M Colpitts, Grant L Hughes, Jason L Rasgon, Tony Nolan, Omar S Akbari, Nelson C Lau
Although mosquitoes are major transmission vectors for pathogenic arboviruses, viral infection has little impact on mosquito health. This immunity is caused in part by mosquito RNA interference (RNAi) pathways that generate antiviral small interfering RNAs (siRNAs) and Piwi-interacting RNAs (piRNAs). RNAi also maintains genome integrity by potently repressing mosquito transposon activity in the germline and soma. However, viral and transposon small RNA regulatory pathways have not been systematically examined together in mosquitoes. Therefore, we developed an integrated mosquito small RNA genomics (MSRG) resource that analyzes the transposon and virus small RNA profiles in mosquito cell cultures and somatic and gonadal tissues across four medically important mosquito species. Our resource captures both somatic and gonadal small RNA expression profiles within mosquito cell cultures, and we report the evolutionary dynamics of a novel Mosquito-Conserved piRNA Cluster Locus (MCpiRCL) made up of satellite DNA repeats. In the larger culicine mosquito genomes we detected highly regular periodicity in piRNA biogenesis patterns coinciding with the expansion of Piwi pathway genes. Finally, our resource enables detection of cross talk between piRNA and siRNA populations in mosquito cells during a response to virus infection. The MSRG resource will aid efforts to dissect and combat the capacity of mosquitoes to tolerate and spread arboviruses.
Antiviral engineeringMosquitoesRNA targeting
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eLife · 2021-Mar-05 · PMID 33666174
Nikolay P Kandul, Junru Liu, Jared B Bennett, John M Marshall, Omar S Akbari
Homing-based gene drives, engineered using CRISPR/Cas9, have been proposed to spread desirable genes throughout populations. However, invasion of such drives can be hindered by the accumulation of resistant alleles. To limit this obstacle, we engineer a confinable population modification home-and-rescue (HomeR) drive in Drosophila targeting an essential gene. In our experiments, resistant alleles that disrupt the target gene function were recessive lethal and therefore disadvantaged. We demonstrate that HomeR can achieve an increase in frequency in population cage experiments, but that fitness costs due to the Cas9 insertion limit drive efficacy. Finally, we conduct mathematical modeling comparing HomeR to contemporary gene drive architectures for population modification over wide ranges of fitness costs, transmission rates, and release regimens. HomeR could potentially be adapted to other species, as a means for safe, confinable, modification of wild populations.
Gene drivesDrosophilaCRISPRGene drive
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Nature communications · 2021-Mar-05 · PMID 33674604
Gerard Terradas, Anna B Buchman, Jared B Bennett, Isaiah Shriner, John M Marshall, Omar S Akbari, Ethan Bier
CRISPR-based gene-drive systems, which copy themselves via gene conversion mediated by the homology-directed repair (HDR) pathway, have the potential to revolutionize vector control. However, mutant alleles generated by the competing non-homologous end-joining (NHEJ) pathway, resistant to Cas9 cleavage, can interrupt the spread of gene-drive elements. We hypothesized that drives targeting genes essential for viability or reproduction also carrying recoded sequences that restore endogenous gene functionality should benefit from dominantly-acting maternal clearance of NHEJ alleles combined with recessive Mendelian culling processes. Here, we test split gene-drive (sGD) systems in Drosophila melanogaster that are inserted into essential genes required for viability (rab5, rab11, prosalpha2) or fertility (spo11). In single generation crosses, sGDs copy with variable efficiencies and display sex-biased transmission. In multigenerational cage trials, sGDs follow distinct drive trajectories reflecting their differential tendencies to induce target chromosome damage and/or lethal/sterile mosaic Cas9-dependent phenotypes, leading to inherently confinable drive outcomes.
Vector controlDrosophilaCRISPR
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Nature communications · 2021-Jun-02 · PMID 34078888
Anna Buchman, Isaiah Shriner, Ting Yang, Junru Liu, Igor Antoshechkin, John M Marshall, Michael W Perry, Omar S Akbari
Engineered reproductive species barriers are useful for impeding gene flow and driving desirable genes into wild populations in a reversible threshold-dependent manner. However, methods to generate synthetic barriers are lacking in advanced eukaryotes. Here, to overcome this challenge, we engineer SPECIES (Synthetic Postzygotic barriers Exploiting CRISPR-based Incompatibilities for Engineering Species), an engineered genetic incompatibility approach, to generate postzygotic reproductive barriers. Using this approach, we create multiple reproductively isolated SPECIES and demonstrate their reproductive isolation and threshold-dependent gene drive capabilities in D. melanogaster. Given the near-universal functionality of CRISPR tools, this approach should be portable to many species, including insect disease vectors in which confinable gene drives could be of great practical utility.
Gene drivesCRISPRGene drive
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Proceedings of the National Academy of Sciences of the United States of America · 2021-Jun-01 · PMID 34031258
Jieyan Chen, Junjie Luo, Yijin Wang, Adishthi S Gurav, Ming Li, Omar S Akbari, Craig Montell
Aedes aegypti spread devastating viruses such as dengue, which causes disease among 100 to 400 million people annually. A potential approach to control mosquito disease vectors is the sterile insect technique (SIT). The strategy involves repeated release of large numbers of sterile males, which reduces insect populations because the sterile males mate and thereby suppress the fertility of females that would otherwise mate with fertile males. While SIT has been successful in suppressing certain agricultural pests, it has been less effective in depressing populations of Ae. aegypti This limitation is in part because of the fitness effects resulting from mutagenizing the mosquitoes nonspecifically. Here, we introduced and characterized the impact on female fertility of an Ae. aegypti mutation that disrupts a gene that is specifically expressed in testes. We used CRISPR/Cas9 to generate a null mutation in the Ae. aegypti β2-tubulin ( B2t ) gene, which eliminates male fertility. When we allowed wild-type females to first mate with B2t mutant males, most of the females did not produce progeny even after being subsequently exposed to wild-type males. We also introduced B2t mutant and wild-type males simultaneously with wild-type females and found that a larger number of B2t mutant males relative to the wild-type males was effective in significantly suppressing female fertility. These results raise the possibility of employing B2t sterile males to improve the efficacy of SIT in suppressing populations of Ae. aegypti through repeated releases and thereby reduce the transmission of viruses by these invasive mosquitoes.
Sterile insect techniquesAntiviral engineeringAedes aegyptiMosquitoesCRISPRSIT
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Nature communications · 2021-Jul-19 · PMID 34282149
Guan-Hong Wang, Stephanie Gamez, Robyn R Raban, John M Marshall, Luke Alphey, Ming Li, Jason L Rasgon, Omar S Akbari
Mosquito-borne diseases, such as dengue and malaria, pose significant global health burdens. Unfortunately, current control methods based on insecticides and environmental maintenance have fallen short of eliminating the disease burden. Scalable, deployable, genetic-based solutions are sought to reduce the transmission risk of these diseases. Pathogen-blocking Wolbachia bacteria, or genome engineering-based mosquito control strategies including gene drives have been developed to address these problems, both requiring the release of modified mosquitoes into the environment. Here, we review the latest developments, notable similarities, and critical distinctions between these promising technologies and discuss their future applications for mosquito-borne disease control.
Gene drivesAntiviral engineeringMosquitoesGene drive
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Genome biology · 2021-Jul-12 · PMID 34253230
Umberto Palatini, Reem A Masri, Luciano V Cosme, Sergey Koren, Françoise Thibaud-Nissen, James K Biedler, Flavia Krsticevic, J Spencer Johnston, Rebecca Halbach, Jacob E Crawford, Igor Antoshechkin, Anna-Bella Failloux, Elisa Pischedda, Michele Marconcini, Jay Ghurye, Arang Rhie, Atashi Sharma, Dmitry A Karagodin, Jeremy Jenrette, Stephanie Gamez, Pascal Miesen, Patrick Masterson, Adalgisa Caccone, Maria V Sharakhova, Zhijian Tu, Philippos A Papathanos, Ronald P Van Rij, Omar S Akbari, Jeffrey Powell, Adam M Phillippy, Mariangela Bonizzoni
No abstract is available from PubMed.
Aedes albopictus
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Journal of visualized experiments : JoVE · 2021-Feb-05 · PMID 33616113
Ruichen Sun, Daniel Brogan, Anna Buchman, Ting Yang, Omar S Akbari
CasRx, a member of the RNA-targeting Cas13 family, is a promising new addition of the CRISPR/Cas technologies in efficient gene transcript reduction with an attractive off-target profile at both cellular and organismal levels. It is recently reported that the CRISPR/CasRx system can be used to achieve ubiquitous and tissue-specific gene transcript reduction in Drosophila melanogaster. This paper details the methods from the recent work, consisting of three parts: 1) ubiquitous in vivo endogenous RNA targeting using a two-component CasRx system; 2) ubiquitous in vivo exogenous RNA targeting using a three-component CasRx system; and 3) tissue-specific in vivo RNA targeting using a three-component CasRx system. The effects of RNA targeting observed include targeted gene specific phenotypic changes, targeted RNA transcript reduction, and occasional lethality phenotypes associated with high expression of CasRx protein and collateral activity. Overall, these results showed that the CasRx system is capable of target RNA transcript reduction at the organismal level in a programmable and efficient manner, demonstrating that in vivo transcriptome targeting, and engineering is feasible and lays the foundation for future in vivo CRISPR-based RNA targeting technologies.
DrosophilaCRISPR
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Scientific reports · 2021-Dec-13 · PMID 34903766
Wei-Liang Liu, Chia-Wei Hsu, Shih-Peng Chan, Pei-Shi Yen, Matthew P Su, Jian-Chiuan Li, Hsing-Han Li, Lie Cheng, Cheng-Kang Tang, Shih-Hsun Ko, Huai-Kuang Tsai, Zing Tsung-Yeh Tsai, Omar S Akbari, Anna-Bella Failloux, Chun-Hong Chen
The areas where dengue virus (DENV) is endemic have expanded rapidly, driven in part by the global spread of Aedes species, which act as disease vectors. DENV replicates in the mosquito midgut and is disseminated to the mosquito's salivary glands for amplification. Thus, blocking virus infection or replication in the tissues of the mosquito may be a viable strategy for reducing the incidence of DENV transmission to humans. Here we used the mariner Mos1 transposase to create an Aedes aegypti line that expresses virus-specific miRNA hairpins capable of blocking DENV replication. These microRNA are driven by the blood-meal-inducible carboxypeptidase A promoter or by the polyubiquitin promoter. The transgenic mosquitoes exhibited significantly lower infection rates and viral titers for most DENV serotypes 7 days after receiving an infectious blood meal. The treatment was also effective at day 14 post infection after a second blood meal had been administered. In viral transmission assay, we found there was significantly reduced transmission in these lines. These transgenic mosquitoes were effective in silencing most of the DENV genome; such an approach may be employed to control a dengue fever epidemic.
Antiviral engineeringSynthetic biologyAedes aegyptiMosquitoesTransgenesis
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Nature communications · 2021-Dec-10 · PMID 34893590
Stephanie Gamez, Duverney Chaverra-Rodriguez, Anna Buchman, Nikolay P Kandul, Stelia C Mendez-Sanchez, Jared B Bennett, Héctor M Sánchez C, Ting Yang, Igor Antoshechkin, Jonny E Duque, Philippos A Papathanos, John M Marshall, Omar S Akbari
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.
Gene drivesSex determinationVector controlSynthetic biologyDrosophilaCRISPR
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The CRISPR journal · 2021-Dec-03 · PMID 34735288
Nikolay P Kandul, Junru Liu, Omar S Akbari
Releases of sterile males are the gold standard for many insect population control programs, and precise sex sorting to remove females prior to male releases is essential to the success of these operations. To advance traditional methods for scaling the generation of sterile males, we previously described a CRISPR-mediated precision-guided sterile insect technique (pgSIT), in which Cas9 and gRNA strains are genetically crossed to generate sterile males for mass release. While effective at generating F 1 sterile males, pgSIT requires a genetic cross between the two parental strains, which requires maintenance and sexing of two strains in a factory. Therefore, to advance pgSIT further by removing this crossing step, here we describe a next-generation temperature-inducible pgSIT (TI-pgSIT) technology and demonstrate its proof-of-concept in Drosophila melanogaster. Importantly, we were able to develop a true breeding strain for TI-pgSIT that eliminates the requirement for sex sorting-a feature that may help further automate production at scale.
Sterile insect techniquesVector controlDrosophilaCRISPRpgSITSIT
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Cells · 2020-Sep-27 · PMID 32992598
Stephanie Gamez, Satyam Srivastav, Omar S Akbari, Nelson C Lau
Animals face the dual threat of virus infections hijacking cellular function and transposons proliferating in germline genomes. For insects, the deeply conserved RNA interference (RNAi) pathways and other chromatin regulators provide an important line of defense against both viruses and transposons. For example, this innate immune system displays adaptiveness to new invasions by generating cognate small RNAs for targeting gene silencing measures against the viral and genomic intruders. However, within the Dipteran clade of insects, Drosophilid fruit flies and Culicids mosquitoes have evolved several unique mechanistic aspects of their RNAi defenses to combat invading transposons and viruses, with the Piwi-piRNA arm of the RNAi pathways showing the greatest degree of novel evolution. Whereas central features of Piwi-piRNA pathways are conserved between Drosophilids and Culicids, multiple lineage-specific innovations have arisen that may reflect distinct genome composition differences and specific ecological and physiological features dividing these two branches of Dipterans. This perspective review focuses on the most recent findings illuminating the Piwi/piRNA pathway distinctions between fruit flies and mosquitoes, and raises open questions that need to be addressed in order to ameliorate human diseases caused by pathogenic viruses that mosquitoes transmit as vectors.
Antiviral engineeringMosquitoesRNA targeting
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Lab animal · 2020-Sep-19 · PMID 32807984
Robyn R Raban, Omar S Akbari
No abstract is available from PubMed.
Mosquitoes
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Global public health · 2020-Oct-26 · PMID 32589115
Cynthia Cheung, Stephanie Gamez, Rebeca Carballar-Lejarazú, Victor Ferman, Váleri N Vásquez, Gerard Terradas, Judy Ishikawa, Cynthia E Schairer, Ethan Bier, John M Marshall, Anthony A James, Omar S Akbari, Cinnamon S Bloss
Information about genetic engineering (GE) for vector control in the United States is disseminated primarily in English, though non-English speakers are equally, and in some geographic regions even more affected by such technologies. Non-English-speaking publics should have equal access to such information, which is especially critical when the technology in question may impact whole communities. We convened an interdisciplinary workgroup to translate previously developed narrated slideshows on gene drive mosquitoes from English into Spanish, reviewing each iteration for scientific accuracy and accessibility to laypeople. Using the finalised stimuli, we conducted five online, chat-based focus groups with Spanish-speaking adults from California. Overall, participants expressed interest in the topic and were able to summarise the information presented in their own words. Importantly, participants asked for clarification and expressed scepticism about the information presented, indicating critical engagement with the material. Through collaboration with Spanish-speaking scientists engaged in the development of GE methods of vector control, we translated highly technical scientific information into Spanish that successfully engaged Spanish-speaking participants in conversations about this topic. In this manuscript, we document the feasibility of consulting Spanish-speaking publics about a complex emerging technology by drawing on the linguistic diversity of the scientific teams developing the technology.
Gene drivesVector controlSynthetic biologyMosquitoesGene drive
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Molecular cell · 2020-Oct-15 · PMID 32949493
Xiang-Ru Shannon Xu, Emily A Bulger, Valentino M Gantz, Carissa Klanseck, Stephanie R Heimler, Ankush Auradkar, Jared B Bennett, Lauren Ashley Miller, Sarah Leahy, Sara Sanz Juste, Anna Buchman, Omar S Akbari, John M Marshall, Ethan Bier
CRISPR-Cas9-based gene drive systems possess the inherent capacity to spread progressively throughout target populations. Here we describe two self-copying (or active) guide RNA-only genetic elements, called e-CHACRs and ERACRs. These elements use Cas9 produced in trans by a gene drive either to inactivate the cas9 transgene (e-CHACRs) or to delete and replace the gene drive (ERACRs). e-CHACRs can be inserted at various genomic locations and carry two or more gRNAs, the first copying the e-CHACR and the second mutating and inactivating the cas9 transgene. Alternatively, ERACRs are inserted at the same genomic location as a gene drive, carrying two gRNAs that cut on either side of the gene drive to excise it. e-CHACRs efficiently inactivate Cas9 and can drive to completion in cage experiments. Similarly, ERACRs, particularly those carrying a recoded cDNA-restoring endogenous gene activity, can drive reliably to fully replace a gene drive. We compare the strengths of these two systems.
Gene drivesCRISPRGene drive
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BMC medical research methodology · 2020-Nov-05 · PMID 33153449
Cynthia E Schairer, Cynthia Triplett, Anna Buchman, Omar S Akbari, Cinnamon S Bloss
While there is wide consensus that the public should be consulted about emerging technology early in development, it is difficult to elicit public opinion about innovations unfamiliar to lay audiences. We sought public input on a program of research on genetic engineering to control mosquito vectors of disease that is led by scientists at the University of California and funded by the U.S. Defense Advanced Research Projects Agency (DARPA). In preparation for this effort, we developed a series of narrated slideshows to prompt responses to the development of gene drive mosquito control strategies among lay people. We describe the development and content of these slideshows and evaluate their ability to elicit discussions among focus group participants.
In developing these materials, we used an iterative process involving input from experts in molecular genetics and vector control. Topics were chosen for their relevance to the goals of the scientists leading the program of research. Significant time was devoted to crafting explanations that would be accessible to uninitiated members of the public but still represent the science accurately. Through qualitative analysis of focus group discussions prompted by the slideshows, we evaluated the success of these slideshows in imparting clear technical information sufficient to inform lay discussion.
The collaboration resulted in a series of four narrated slideshows that were used to anchor discussions in online focus groups. Many participants described the slideshows as interesting and informative, while also raising concerns and possible risks that were not directly addressed in the material presented. Open-ended comments from participants suggest that the slideshows inspired critical questions, reflection, and conversation about genetically engineered and gene drive mosquitoes. After the final and most technically complex slideshow, however, some respondents made comments suggestive of overwhelm or confusion.
Our narrated slideshows prompted engaged conversations about genetically engineered mosquitoes among members of the public who were generally naïve to this technology. Narrated slideshows may serve as viable and useful tools for future public engagement on other controversial emerging medical and public health technologies.
Gene drivesVector controlSynthetic biologyMosquitoesGene drive
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Journal of visualized experiments : JoVE · 2020-May-24 · PMID 32510506
Michelle Bui, Ming Li, Robyn R Raban, Nannan Liu, Omar S Akbari
Culex quinquefasciatus is a vector of a diverse range of vector-borne diseases such as avian malaria, West Nile virus (WNV), Japanese encephalitis, Eastern equine encephalitis, lymphatic filariasis, and Saint Louis encephalitis. Notably, avian malaria has played a major role in the extinction of numerous endemic island bird species, while WNV has become an important vector-borne disease in the United States. To gain further insight into C. quinquefasciatus biology and expand their genetic control toolbox, we need to develop more efficient and affordable methods for genome engineering in this species. However, some biological traits unique to Culex mosquitoes, particularly their egg rafts, have made it difficult to perform microinjection procedures required for genome engineering. To address these challenges, we have developed an optimized embryo microinjection protocol that focuses on mitigating the technical obstacles associated with the unique characteristics of Culex mosquitoes. These procedures demonstrate optimized methods for egg collection, egg raft separation and other handling procedures essential for successful microinjection in C. quinquefasciatus. When coupled with the CRISPR/Cas9 genome editing technology, these procedures allow us to achieve site-specific, efficient and heritable germline mutations, which are required to perform advanced genome engineering and develop genetic control technologies in this important, but currently understudied, disease vector.
Antiviral engineeringCulexMosquitoesCRISPR
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BMC biology · 2020-May-12 · PMID 32398005
Héctor M Sánchez C, Jared B Bennett, Sean L Wu, Gordana Rašić, Omar S Akbari, John M Marshall
The discovery of CRISPR-based gene editing and its application to homing-based gene drive systems has been greeted with excitement, for its potential to control mosquito-borne diseases on a wide scale, and concern, for the invasiveness and potential irreversibility of a release. Gene drive systems that display threshold-dependent behavior could potentially be used during the trial phase of this technology, or when localized control is otherwise desired, as simple models predict them to spread into partially isolated populations in a confineable manner, and to be reversible through releases of wild-type organisms. Here, we model hypothetical releases of two recently engineered threshold-dependent gene drive systems-reciprocal chromosomal translocations and a form of toxin-antidote-based underdominance known as UD MEL -to explore their ability to be confined and remediated.
We simulate releases of Aedes aegypti, the mosquito vector of dengue, Zika, and other arboviruses, in Yorkeys Knob, a suburb of Cairns, Australia, where previous biological control interventions have been undertaken on this species. We monitor spread to the neighboring suburb of Trinity Park to assess confinement. Results suggest that translocations could be introduced on a suburban scale, and remediated through releases of non-disease-transmitting male mosquitoes with release sizes on the scale of what has been previously implemented. UD MEL requires fewer releases to introduce, but more releases to remediate, including of females capable of disease transmission. Both systems are expected to be confineable to the release site; however, spillover of translocations into neighboring populations is less likely.
Our analysis supports the use of translocations as a threshold-dependent drive system capable of spreading disease-refractory genes into Ae. aegypti populations in a confineable and reversible manner. It also highlights increased release requirements when incorporating life history and population structure into models. As the technology nears implementation, further ecological work will be essential to enhance model predictions in preparation for field trials.
Gene drivesAntiviral engineeringAedes aegyptiMosquitoesCRISPRGene drive
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G3 (Bethesda, Md.) · 2020-Mar-05 · PMID 31964684
Stephanie Gamez, Igor Antoshechkin, Stelia C Mendez-Sanchez, Omar S Akbari
Aedes albopictus mosquitoes are important vectors for a number of human pathogens including the Zika, dengue, and chikungunya viruses. Capable of displacing Aedes aegypti populations, this mosquito adapts to cooler environments which increases its geographical range and transmission potential. There are limited control strategies for Aedes albopictus mosquitoes which is likely attributed to the lack of comprehensive biological studies on this emerging vector. To fill this void, here using RNAseq we characterized Aedes albopictus mRNA expression profiles at 34 distinct time points throughout development providing the first high-resolution comprehensive view of the developmental transcriptome of this worldwide human disease vector. This enabled us to identify several patterns of shared gene expression among tissues as well as sex-specific expression patterns. To illuminate the similarities and differences with Aedes aegypti , a related human disease vector, we also performed a comparative analysis between the two developmental transcriptomes, identifying life stages where the two species exhibit similar and distinct gene expression patterns. These findings provide insights into the similarities and differences between Aedes albopictus and Aedes aegypti mosquito biology. In summary, the results generated from this study should form the basis for future investigations on the biology of Aedes albopictus and provide a gold mine resource for the development of transgene-based vector control strategies.
Antiviral engineeringVector controlAedes aegyptiAedes albopictusMosquitoes
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The CRISPR journal · 2020-Jun-26 · PMID 32584145
Anna B Buchman, Dan J Brogan, Ruichen Sun, Ting Yang, Patrick D Hsu, Omar S Akbari
CRISPR-Cas genome editing technologies have revolutionized the fields of functional genetics and genome engineering, but with the recent discovery and optimization of RNA-targeting Cas ribonucleases, we may soon see a similar revolution in the study of RNA function and transcriptome engineering. However, to date, successful proof of principle for Cas ribonuclease RNA targeting in eukaryotic systems has been limited. Only recently has successful modification of RNA expression by a Cas ribonuclease been demonstrated in animal embryos. This previous work, however, did not evaluate endogenous expression of Cas ribonucleases and only focused on function in early developmental stages. A more comprehensive evaluation of this technology is needed to assess its potential impact. Here we report on our efforts to develop a programmable platform for RNA targeting using a Cas ribonuclease, CasRx, in the model organism Drosophila melanogaster . By genetically encoding CasRx in flies, we demonstrate moderate transcript targeting of known phenotypic genes in addition to unexpected toxicity and lethality. We also report on the off-target effects following on-target transcript cleavage by CasRx. Taken together, our results present the current state and limitations of a genetically encoded programmable RNA-targeting Cas system in Drosophila melanogaster , paving the way for future optimization of the system.
DrosophilaCRISPR
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eLife · 2020-Jan-21 · PMID 31960794
Ming Li, Ting Yang, Nikolay P Kandul, Michelle Bui, Stephanie Gamez, Robyn Raban, Jared Bennett, Héctor M Sánchez C, Gregory C Lanzaro, Hanno Schmidt, Yoosook Lee, John M Marshall, Omar S Akbari
Aedes aegypti is the principal mosquito vector for many arboviruses that increasingly infect millions of people every year. With an escalating burden of infections and the relative failure of traditional control methods, the development of innovative control measures has become of paramount importance. The use of gene drives has sparked significant enthusiasm for genetic control of mosquitoes; however, no such system has been developed in Ae. aegypti . To fill this void, here we develop several CRISPR-based split gene drives for use in this vector. With cleavage rates up to 100% and transmission rates as high as 94%, mathematical models predict that these systems could spread anti-pathogen effector genes into wild populations in a safe, confinable and reversible manner appropriate for field trials and effective for controlling disease. These findings could expedite the development of effector-linked gene drives that could safely control wild populations of Ae. aegypti to combat local pathogen transmission.
Gene drivesAntiviral engineeringAedes aegyptiMosquitoesCRISPRGene drive
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PLoS pathogens · 2020-Jan-16 · PMID 31945137
Anna Buchman, Stephanie Gamez, Ming Li, Igor Antoshechkin, Hsing-Han Li, Hsin-Wei Wang, Chun-Hong Chen, Melissa J Klein, Jean-Bernard Duchemin, James E Crowe, Prasad N Paradkar, Omar S Akbari
With dengue virus (DENV) becoming endemic in tropical and subtropical regions worldwide, there is a pressing global demand for effective strategies to control the mosquitoes that spread this disease. Recent advances in genetic engineering technologies have made it possible to create mosquitoes with reduced vector competence, limiting their ability to acquire and transmit pathogens. Here we describe the development of Aedes aegypti mosquitoes synthetically engineered to impede vector competence to DENV. These mosquitoes express a gene encoding an engineered single-chain variable fragment derived from a broadly neutralizing DENV human monoclonal antibody and have significantly reduced viral infection, dissemination, and transmission rates for all four major antigenically distinct DENV serotypes. Importantly, this is the first engineered approach that targets all DENV serotypes, which is crucial for effective disease suppression. These results provide a compelling route for developing effective genetic-based DENV control strategies, which could be extended to curtail other arboviruses.
Antiviral engineeringSynthetic biologyAedes aegyptiMosquitoes
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Proceedings of the National Academy of Sciences of the United States of America · 2020-Jan-07 · PMID 31871198
Chloé Lahondère, Clément Vinauger, Ryo P Okubo, Gabriella H Wolff, Jeremy K Chan, Omar S Akbari, Jeffrey A Riffell
Mosquitoes are important vectors of disease and require sources of carbohydrates for reproduction and survival. Unlike host-related behaviors of mosquitoes, comparatively less is understood about the mechanisms involved in nectar-feeding decisions, or how this sensory information is processed in the mosquito brain. Here we show that Aedes spp. mosquitoes, including Aedes aegypti , are effective pollinators of the Platanthera obtusata orchid, and demonstrate this mutualism is mediated by the orchid's scent and the balance of excitation and inhibition in the mosquito's antennal lobe (AL). The P. obtusata orchid emits an attractive, nonanal-rich scent, whereas related Platanthera species-not visited by mosquitoes-emit scents dominated by lilac aldehyde. Calcium imaging experiments in the mosquito AL revealed that nonanal and lilac aldehyde each respectively activate the LC2 and AM2 glomerulus, and remarkably, the AM2 glomerulus is also sensitive to N,N-diethyl-meta-toluamide (DEET), a mosquito repellent. Lateral inhibition between these 2 glomeruli reflects the level of attraction to the orchid scents. Whereas the enriched nonanal scent of P. obtusata activates the LC2 and suppresses AM2, the high level of lilac aldehyde in the other orchid scents inverts this pattern of glomerular activity, and behavioral attraction is lost. These results demonstrate the ecological importance of mosquitoes beyond operating as disease vectors and open the door toward understanding the neural basis of mosquito nectar-seeking behaviors.
Aedes aegyptiMosquitoes
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The Journal of experimental biology · 2020-Feb-07 · PMID 32034041
Robyn R Raban, John M Marshall, Omar S Akbari
Vector-borne diseases, such as dengue, Zika and malaria, are a major cause of morbidity and mortality worldwide. These diseases have proven difficult to control and currently available management tools are insufficient to eliminate them in many regions. Gene drives have the potential to revolutionize vector-borne disease control. This suite of technologies has advanced rapidly in recent years as a result of the availability of new, more efficient gene editing technologies. Gene drives can favorably bias the inheritance of a linked disease-refractory gene, which could possibly be exploited (i) to generate a vector population incapable of transmitting disease or (ii) to disrupt an essential gene for viability or fertility, which could eventually eliminate a population. Importantly, gene drives vary in characteristics such as their transmission efficiency, confinability and reversibility, and their potential to develop resistance to the drive mechanism. Here, we discuss recent advancements in the gene drive field, and contrast the benefits and limitations of a variety of technologies, as well as approaches to overcome these limitations. We also discuss the current state of each gene drive technology and the technical considerations that need to be addressed on the pathway to field implementation. While there are still many obstacles to overcome, recent progress has brought us closer than ever before to genetic-based vector modification as a tool to support vector-borne disease elimination efforts worldwide.
Gene drivesAntiviral engineeringVector controlGene drive
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G3 (Bethesda, Md.) · 2020-Feb-06 · PMID 31882406
Nikolay P Kandul, Junru Liu, Anna Buchman, Valentino M Gantz, Ethan Bier, Omar S Akbari
Homing based gene drives (HGD) possess the potential to spread linked cargo genes into natural populations and are poised to revolutionize population control of animals. Given that host encoded genes have been identified that are important for pathogen transmission, targeting these genes using guide RNAs as cargo genes linked to drives may provide a robust method to prevent disease transmission. However, effectiveness of the inclusion of additional guide RNAs that target separate genes has not been thoroughly explored. To test this approach, we generated a split-HGD in Drosophila melanogaster that encoded a drive linked effector consisting of a second gRNA engineered to target a separate host-encoded gene, which we term a gRNA-mediated effector (GME). This design enabled us to assess homing and knockout efficiencies of two target genes simultaneously, and also explore the timing and tissue specificity of Cas9 expression on cleavage/homing rates. We demonstrate that inclusion of a GME can result in high efficiency of disruption of both genes during super-Mendelian propagation of split-HGD. Furthermore, both genes were knocked out one generation earlier than expected indicating the robust somatic expression of Cas9 driven by Drosophila germline-limited promoters. We also assess the efficiency of 'shadow drive' generated by maternally deposited Cas9 protein and accumulation of drive-induced resistance alleles along multiple generations, and discuss design principles of HGD that could mitigate the accumulation of resistance alleles while incorporating a GME.
Gene drivesVector controlDrosophilaCRISPRGene drive
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Science (New York, N.Y.) · 2020-Dec-18 · PMID 33335055
Kanya C Long, Luke Alphey, George J Annas, Cinnamon S Bloss, Karl J Campbell, Jackson Champer, Chun-Hong Chen, Amit Choudhary, George M Church, James P Collins, Kimberly L Cooper, Jason A Delborne, Owain R Edwards, Claudia I Emerson, Kevin Esvelt, Sam Weiss Evans, Robert M Friedman, Valentino M Gantz, Fred Gould, Sarah Hartley, Elizabeth Heitman, Janet Hemingway, Hirotaka Kanuka, Jennifer Kuzma, James V Lavery, Yoosook Lee, Marce Lorenzen, Jeantine E Lunshof, John M Marshall, Philipp W Messer, Craig Montell, Kenneth A Oye, Megan J Palmer, Philippos Aris Papathanos, Prasad N Paradkar, Antoinette J Piaggio, Jason L Rasgon, Gordana Rašić, Larisa Rudenko, J Royden Saah, Maxwell J Scott, Jolene T Sutton, Adam E Vorsino, Omar S Akbari
No abstract is available from PubMed.
Gene drivesGene drive
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Proceedings of the National Academy of Sciences of the United States of America · 2020-Dec-08 · PMID 33208534
Luke S Alphey, Andrea Crisanti, Filippo Fil Randazzo, Omar S Akbari
No abstract is available from PubMed.
Gene drivesGene drive
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Journal of visualized experiments : JoVE · 2020-Dec-04 · PMID 33346197
Elena Dalla Benetta, Duverney Chaverra-Rodriguez, Jason L Rasgon, Omar S Akbari
The jewel wasp, Nasonia vitripennis, has become an efficient model system to study epigenetics of haplo-diploid sex determination, B-chromosome biology, host-symbiont interactions, speciation, and venom synthesis. Despite the availability of several molecular tools, including CRISPR/Cas9, functional genetic studies are still limited in this organism. The major limitation of applying CRISPR/Cas9 technology in N. vitripennis stems from the challenges of embryonic microinjections. Injections of embryos are particularly difficult in this organism and in general in many parasitoid wasps, due to small embryo size and the requirement of a host pupa for embryonic development. To address these challenges, Cas9 ribonucleoprotein complex delivery into female ovaries by adult injection, rather than embryonic microinjection, was optimized, resulting in both somatic and heritable germline edits. The injection procedures were optimized in pupae and female wasps using either ReMOT Control (Receptor-Mediated Ovary Transduction of Cargo) or BAPC (Branched Amphiphilic Peptide Capsules). These methods are shown to be effective alternatives to embryo injection, enabling site-specific and heritable germline mutations.
Sex determinationCRISPRRNA targeting
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PLoS neglected tropical diseases · 2020-Dec-03 · PMID 33270627
David Navarro-Payá, Ilona Flis, Michelle A E Anderson, Philippa Hawes, Ming Li, Omar S Akbari, Sanjay Basu, Luke Alphey
Aedes aegypti Act4 is a paralog of the Drosophila melanogaster indirect flight muscle actin gene Act88F. Act88F has been shown to be haploinsufficient for flight in both males and females (amorphic mutants are dominant). Whereas Act88F is expressed in indirect flight muscles of both males and females, expression of Act4 is substantially female-specific. We therefore used CRISPR/Cas9 and homology directed repair to examine the phenotype of Act4 mutants in two Culicine mosquitoes, Aedes aegypti and Culex quinquefasciatus. A screen for dominant female-flightless mutants in Cx. quinquefasciatus identified one such mutant associated with a six base pair deletion in the CxAct4 coding region. A similar screen in Ae. aegypti identified no dominant mutants. Disruption of the AeAct4 gene by homology-dependent insertion of a fluorescent protein marker cassette gave a recessive female-flightless phenotype in Ae. aegypti. Reproducing the six-base deletion from Cx. quinquefasciatus in Ae. aegypti using oligo-directed mutagenesis generated dominant female-flightless mutants and identified additional dominant female-flightless mutants with other in-frame insertions or deletions. Our data indicate that loss of function mutations in the AeAct4 gene are recessive but that short in-frame deletions produce dominant-negative versions of the AeAct4 protein that interfere with flight muscle function. This makes Act4 an interesting candidate for genetic control methods, particularly population-suppression gene drives targeting female viability/fertility.
Gene drivesAedes aegyptiCulexDrosophilaMosquitoesCRISPR
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Genome biology · 2020-Aug-26 · PMID 32847630
Umberto Palatini, Reem A Masri, Luciano V Cosme, Sergey Koren, Françoise Thibaud-Nissen, James K Biedler, Flavia Krsticevic, J Spencer Johnston, Rebecca Halbach, Jacob E Crawford, Igor Antoshechkin, Anna-Bella Failloux, Elisa Pischedda, Michele Marconcini, Jay Ghurye, Arang Rhie, Atashi Sharma, Dmitry A Karagodin, Jeremy Jenrette, Stephanie Gamez, Pascal Miesen, Patrick Masterson, Adalgisa Caccone, Maria V Sharakhova, Zhijian Tu, Philippos A Papathanos, Ronald P Van Rij, Omar S Akbari, Jeffrey Powell, Adam M Phillippy, Mariangela Bonizzoni
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.
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.
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.
Antiviral engineeringVector controlAedes albopictusMosquitoes
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Nature communications · 2020-Apr-30 · PMID 32355156
Nikolay P Kandul, Junru Liu, Alexander D Hsu, Bruce A Hay, Omar S Akbari
Here, we describe a drug-inducible genetic system for insect sex-separation that demonstrates proof-of-principle for positive sex selection in D. melanogaster. The system exploits the toxicity of commonly used broad-spectrum antibiotics geneticin and puromycin to kill the non-rescued sex. Sex-specific rescue is achieved by inserting sex-specific introns into the coding sequences of antibiotic-resistance genes. When raised on geneticin-supplemented food, the sex-sorter line establishes 100% positive selection for female progeny, while the food supplemented with puromycin positively selects 100% male progeny. Since the described system exploits conserved sex-specific splicing mechanisms and reagents, it has the potential to be adaptable to other insect species of medical and agricultural importance.
View paper record →laboratoryAbstract onlycorrected
PLoS pathogens · 2020-Apr-29 · PMID 32348355
Anna Buchman, Stephanie Gamez, Ming Li, Igor Antoshechkin, Hsing-Han Li, Hsin-Wei Wang, Chun-Hong Chen, Melissa J Klein, Jean-Bernard Duchemin, James E Crowe, Prasad N Paradkar, Omar S Akbari
[This corrects the article DOI: 10.1371/journal.ppat.1008103.].
Antiviral engineeringMosquitoes
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Science advances · 2020-Apr-03 · PMID 32284986
Elena Dalla Benetta, Igor Antoshechkin, Ting Yang, Hoa Quang My Nguyen, Patrick M Ferree, Omar S Akbari
Numerous plants and animals harbor selfish B chromosomes that "drive" or transmit themselves at super-Mendelian frequencies, despite long-term fitness costs to the organism. Currently, it is unknown how B chromosome drive is mediated, and whether B-gene expression plays a role. We used modern sequencing technologies to analyze the fine-scale sequence composition and expression of paternal sex ratio (PSR), a B chromosome in the jewel wasp Nasonia vitripennis . PSR causes female-to-male conversion by destroying the sperm's hereditary material in young embryos to drive. Using RNA interference, we demonstrate that testis-specific expression of a PSR-linked gene, named haploidizer , facilitates this genome elimination-and-sex conversion effect. haploidizer encodes a putative protein with a DNA binding domain, suggesting a functional link with the sperm-derived chromatin.
Sex determinationRNA targeting
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Lancet (London, England) · 2019-Sep-21 · PMID 31511196
Richard G A Feachem, Ingrid Chen, Omar Akbari, Amelia Bertozzi-Villa, Samir Bhatt, Fred Binka, Maciej F Boni, Caroline Buckee, Joseph Dieleman, Arjen Dondorp, Alex Eapen, Neelam Sekhri Feachem, Scott Filler, Peter Gething, Roly Gosling, Annie Haakenstad, Kelly Harvard, Arian Hatefi, Dean Jamison, Kate E Jones, Corine Karema, Richard Nchabi Kamwi, Altaf Lal, Erika Larson, Margaret Lees, Neil F Lobo, Angela E Micah, Bruno Moonen, Gretchen Newby, Xiao Ning, Muhammad Pate, Martha Quiñones, Michelle Roh, Ben Rolfe, Dennis Shanks, Balbir Singh, Kenneth Staley, James Tulloch, Jennifer Wegbreit, Hyun Ju Woo, Winnie Mpanju-Shumbusho
No abstract is available from PubMed.
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Nature communications · 2019-Sep-02 · PMID 31477702
Nikolay P Kandul, Junru Liu, Hector M Sanchez C, Sean L Wu, John M Marshall, Omar S Akbari
No abstract is available from PubMed.
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PLoS neglected tropical diseases · 2019-Nov-25 · PMID 31765377
Cynthia E Schairer, Riley Taitingfong, Omar S Akbari, Cinnamon S Bloss
Despite broad consensus on the importance of community and stakeholder engagement (CSE) for guiding the development, regulation, field testing, and deployment of emerging vector control technologies (such as genetically engineered insects), the types of activities pursued have varied widely, as have the outcomes. We looked to previous CSE efforts for clarity about appropriate methods and goals. Our analysis yielded a typology of CSE, and related vocabulary, that describes distinctions that funders, organizers, and scholars should make when proposing or evaluating CSE.
We compiled available formal documentation of CSE projects, starting with projects mentioned in interviews with 17 key informants. Major features of these examples, including the initiators, target groups, timing, goals, and methods were identified using qualitative coding. Based on these examples, subcategories were developed for a subset of features and applied to the identified cases of CSE in the documents. Co-occurrence of subcategorized features was examined for patterns.
We identified 14 documented examples CSE projects, which were comprised of 28 distinct CSE activities. We found no clear patterns with respect to timing. However, we found that grouping examples according to whether initiators or targets could enact the immediate desired outcome could help to clarify relationships between goals, methods, and targets.
Based on this analysis, we propose a typology that distinguishes three categories of CSE: engagement to inquire -where initiators are empowered to act on information collected through engagement with target groups; engagement to influence -where initiators engage to affect the actions of already-empowered target groups; and engagement to involve -where initiators engage to delegate authority to target groups. The proposed typology can serve as a guide for establishing the goals, identifying appropriate methods, and evaluating and reporting CSE projects by directing attention to important questions to be asked well before determining who to engage and how.
Vector control
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BMC neuroscience · 2019-Jun-17 · PMID 31208328
Michelle Bui, Jennifer Shyong, Eleanor K Lutz, Ting Yang, Ming Li, Kenneth Truong, Ryan Arvidson, Anna Buchman, Jeffrey A Riffell, Omar S Akbari
The mosquito Aedes aegypti has a wide variety of sensory pathways that have supported its success as a species as well as a highly competent vector of numerous debilitating infectious pathogens. Investigations into mosquito sensory systems and their effects on behavior are valuable resources for the advancement of mosquito control strategies. Numerous studies have elucidated key aspects of mosquito sensory systems, however there remains critical gaps within the field. In particular, compared to that of the adult form, there has been a lack of studies directed towards the immature life stages. Additionally, although numerous studies have pinpointed specific sensory receptors as well as responding motor outputs, there has been a lack of studies able to monitor both concurrently.
To begin filling aforementioned gaps, here we engineered Ae. aegypti to ubiquitously express a genetically encoded calcium indicator, GCaMP6s. Using this strain, combined with advanced microscopy, we simultaneously measured live stimulus-evoked calcium responses in both neuronal and muscle cells with a wide spatial range and resolution.
By coupling in vivo live calcium imaging with behavioral assays we were able to gain functional insights into how stimulus-evoked neural and muscle activities are represented, modulated, and transformed in mosquito larvae enabling us to elucidate mosquito sensorimotor properties important for life-history-specific foraging strategies.
Aedes aegyptiMosquitoes
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Nature communications · 2019-Jan-08 · PMID 30622266
Nikolay P Kandul, Junru Liu, Hector M Sanchez C, Sean L Wu, John M Marshall, Omar S Akbari
The sterile insect technique (SIT) is an environmentally safe and proven technology to suppress wild populations. To further advance its utility, a novel CRISPR-based technology termed precision guided SIT (pgSIT) is described. PgSIT mechanistically relies on a dominant genetic technology that enables simultaneous sexing and sterilization, facilitating the release of eggs into the environment ensuring only sterile adult males emerge. Importantly, for field applications, the release of eggs will eliminate burdens of manually sexing and sterilizing males, thereby reducing overall effort and increasing scalability. Here, to demonstrate efficacy, we systematically engineer multiple pgSIT systems in Drosophila which consistently give rise to 100% sterile males. Importantly, we demonstrate that pgSIT-generated sterile males are fit and competitive. Using mathematical models, we predict pgSIT will induce substantially greater population suppression than can be achieved by currently-available self-limiting suppression technologies. Taken together, pgSIT offers to potentially transform our ability to control insect agricultural pests and disease vectors.
Sterile insect techniquesVector controlDrosophilaCRISPRpgSITSIT
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Proceedings of the National Academy of Sciences of the United States of America · 2019-Feb-26 · PMID 30723148
Anna Buchman, Stephanie Gamez, Ming Li, Igor Antoshechkin, Hsing-Han Li, Hsin-Wei Wang, Chun-Hong Chen, Melissa J Klein, Jean-Bernard Duchemin, Prasad N Paradkar, Omar S Akbari
Recent Zika virus (ZIKV) outbreaks have highlighted the necessity for development of novel vector control strategies to combat arboviral transmission, including genetic versions of the sterile insect technique, artificial infection with Wolbachia to reduce population size and/or vectoring competency, and gene drive-based methods. Here, we describe the development of mosquitoes synthetically engineered to impede vector competence to ZIKV. We demonstrate that a polycistronic cluster of engineered synthetic small RNAs targeting ZIKV is expressed and fully processed in Aedes aegypti , ensuring the formation of mature synthetic small RNAs in the midgut where ZIKV resides in the early stages of infection. Critically, we demonstrate that engineered Ae. aegypti mosquitoes harboring the anti-ZIKV transgene have significantly reduced viral infection, dissemination, and transmission rates of ZIKV. Taken together, these compelling results provide a promising path forward for development of effective genetic-based ZIKV control strategies, which could potentially be extended to curtail other arboviruses.
Sterile insect techniquesGene drivesAntiviral engineeringVector controlSynthetic biologyAedes aegypti
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Genes · 2019-Feb-08 · PMID 30744010
Elena Dalla Benetta, Omar S Akbari, Patrick M Ferree
B chromosomes are enigmatic heritable elements found in the genomes of numerous plant and animal species. Contrary to their broad distribution, most B chromosomes are non-essential. For this reason, they are regarded as genome parasites. In order to be stably transmitted through generations, many B chromosomes exhibit the ability to "drive", i.e., they transmit themselves at super-Mendelian frequencies to progeny through directed interactions with the cell division apparatus. To date, very little is understood mechanistically about how B chromosomes drive, although a likely scenario is that expression of B chromosome sequences plays a role. Here, we highlight a handful of previously identified B chromosome sequences, many of which are repetitive and non-coding in nature, that have been shown to be expressed at the transcriptional level. We speculate on how each type of expressed sequence could participate in B chromosome drive based on known functions of RNA in general chromatin- and chromosome-related processes. We also raise some challenges to functionally testing these possible roles, a goal that will be required to more fully understand whether and how B chromosomes interact with components of the cell for drive and transmission .
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Current biology : CB · 2019-Aug-05 · PMID 31327719
Clément Vinauger, Floris Van Breugel, Lauren T Locke, Kennedy K S Tobin, Michael H Dickinson, Adrienne L Fairhall, Omar S Akbari, Jeffrey A Riffell
Mosquitoes rely on the integration of multiple sensory cues, including olfactory, visual, and thermal stimuli, to detect, identify, and locate their hosts [1-4]. Although we increasingly know more about the role of chemosensory behaviors in mediating mosquito-host interactions [1], the role of visual cues is comparatively less studied [3], and how the combination of olfactory and visual information is integrated in the mosquito brain remains unknown. In the present study, we used a tethered-flight light-emitting diode (LED) arena, which allowed for quantitative control over the stimuli, and a control theoretic model to show that CO 2 modulates mosquito steering responses toward vertical bars. To gain insight into the neural basis of this olfactory and visual coupling, we conducted two-photon microscopy experiments in a new GCaMP6s-expressing mosquito line. Imaging revealed that neuropil regions within the lobula exhibited strong responses to objects, such as a bar, but showed little response to a large-field motion. Approximately 20% of the lobula neuropil we imaged were modulated when CO 2 preceded the presentation of a moving bar. By contrast, responses in the antennal (olfactory) lobe were not modulated by visual stimuli presented before or after an olfactory stimulus. Together, our results suggest that asymmetric coupling between these sensory systems provides enhanced steering responses to discrete objects.
Aedes aegyptiMosquitoes
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Frontiers in genetics · 2019-10-30 · PMID 31737050
John M Marshall, Robyn R Raban, Nikolay P Kandul, Jyotheeswara R Edula, Tomás M León, Omar S Akbari
While efforts to control malaria with available tools have stagnated, and arbovirus outbreaks persist around the globe, the advent of clustered regularly interspaced short palindromic repeat (CRISPR)-based gene editing has provided exciting new opportunities for genetics-based strategies to control these diseases. In one such strategy, called "population replacement", mosquitoes, and other disease vectors are engineered with effector genes that render them unable to transmit pathogens. These effector genes can be linked to "gene drive" systems that can bias inheritance in their favor, providing novel opportunities to replace disease-susceptible vector populations with disease-refractory ones over the course of several generations. While promising for the control of vector-borne diseases on a wide scale, this sets up an evolutionary tug-of-war between the introduced effector genes and the pathogen. Here, we review the disease-refractory genes designed to date to target Plasmodium falciparum malaria transmitted by Anopheles gambiae , and arboviruses transmitted by Aedes aegypti , including dengue serotypes 2 and 3, chikungunya, and Zika viruses. We discuss resistance concerns for these effector genes, and genetic approaches to prevent parasite and viral escape variants. One general approach is to increase the evolutionary hurdle required for the pathogen to evolve resistance by attacking it at multiple sites in its genome and/or multiple stages of development. Another is to reduce the size of the pathogen population by other means, such as with vector control and antimalarial drugs. We discuss lessons learned from the evolution of resistance to antimalarial and antiviral drugs and implications for the management of resistance after its emergence. Finally, we discuss the target product profile for population replacement strategies for vector-borne disease control. This differs between early phase field trials and wide-scale disease control. In the latter case, the demands on effector gene efficacy are great; however, with new possibilities ushered in by CRISPR-based gene editing, and when combined with surveillance, monitoring, and rapid management of pathogen resistance, the odds are increasingly favoring effector genes in the upcoming evolutionary tug-of-war.
Gene drivesAntiviral engineeringVector controlAedes aegyptiAnophelesMosquitoes
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Nature · 2018-Nov-14 · PMID 30429615
Benjamin J Matthews, Olga Dudchenko, Sarah B Kingan, Sergey Koren, Igor Antoshechkin, Jacob E Crawford, William J Glassford, Margaret Herre, Seth N Redmond, Noah H Rose, Gareth D Weedall, Yang Wu, Sanjit S Batra, Carlos A Brito-Sierra, Steven D Buckingham, Corey L Campbell, Saki Chan, Eric Cox, Benjamin R Evans, Thanyalak Fansiri, Igor Filipović, Albin Fontaine, Andrea Gloria-Soria, Richard Hall, Vinita S Joardar, Andrew K Jones, Raissa G G Kay, Vamsi K Kodali, Joyce Lee, Gareth J Lycett, Sara N Mitchell, Jill Muehling, Michael R Murphy, Arina D Omer, Frederick A Partridge, Paul Peluso, Aviva Presser Aiden, Vidya Ramasamy, Gordana Rašić, Sourav Roy, Karla Saavedra-Rodriguez, Shruti Sharan, Atashi Sharma, Melissa Laird Smith, Joe Turner, Allison M Weakley, Zhilei Zhao, Omar S Akbari, William C Black, Han Cao, Alistair C Darby, Catherine A Hill, J Spencer Johnston, Terence D Murphy, Alexander S Raikhel, David B Sattelle, Igor V Sharakhov, Bradley J White, Li Zhao, Erez Lieberman Aiden, Richard S Mann, Louis Lambrechts, Jeffrey R Powell, Maria V Sharakhova, Zhijian Tu, Hugh M Robertson, Carolyn S McBride, Alex R Hastie, Jonas Korlach, Daniel E Neafsey, Adam M Phillippy, Leslie B Vosshall
Female Aedes aegypti mosquitoes infect more than 400 million people each year with dangerous viral pathogens including dengue, yellow fever, Zika and chikungunya. Progress in understanding the biology of mosquitoes and developing the tools to fight them has been slowed by the lack of a high-quality genome assembly. Here we combine diverse technologies to produce the markedly improved, fully re-annotated AaegL5 genome assembly, and demonstrate how it accelerates mosquito science. We anchored physical and cytogenetic maps, doubled the number of known chemosensory ionotropic receptors that guide mosquitoes to human hosts and egg-laying sites, provided further insight into the size and composition of the sex-determining M locus, and revealed copy-number variation among glutathione S-transferase genes that are important for insecticide resistance. Using high-resolution quantitative trait locus and population genomic analyses, we mapped new candidates for dengue vector competence and insecticide resistance. AaegL5 will catalyse new biological insights and intervention strategies to fight this deadly disease vector.
Antiviral engineeringVector controlAedes aegyptiMosquitoes
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ACS synthetic biology · 2018-May-18 · PMID 29608276
Anna B Buchman, Tobin Ivy, John M Marshall, Omar S Akbari, Bruce A Hay
Replacement of wild insect populations with transgene-bearing individuals unable to transmit disease or survive under specific environmental conditions using gene drive provides a self-perpetuating method of disease prevention. Mechanisms that require the gene drive element and linked cargo to exceed a high threshold frequency in order for spread to occur are attractive because they offer several points of control: they bring about local, but not global population replacement; and transgenes can be eliminated by reintroducing wildtypes into the population so as to drive the frequency of transgenes below the threshold frequency required for drive. Reciprocal chromosome translocations were proposed as a tool for bringing about high threshold population replacement in 1940 and 1968. However, translocations able to achieve this goal have only been reported once, in the spider mite Tetranychus urticae, a haplo-diploid species in which there is strong selection in haploid males for fit homozygotes. We report the creation of engineered translocation-bearing strains of Drosophila melanogaster, generated through targeted chromosomal breakage and homologous recombination. These strains drive high threshold population replacement in laboratory populations. While it remains to be shown that engineered translocations can bring about population replacement in wild populations, these observations suggest that further exploration of engineered translocations as a tool for controlled population replacement is warranted.
Gene drivesDrosophilaGene drive
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Proceedings of the National Academy of Sciences of the United States of America · 2018-May-01 · PMID 29666236
Anna Buchman, John M Marshall, Dennis Ostrovski, Ting Yang, Omar S Akbari
Synthetic gene drive systems possess enormous potential to replace, alter, or suppress wild populations of significant disease vectors and crop pests; however, their utility in diverse populations remains to be demonstrated. Here, we report the creation of a synthetic Medea gene drive system in a major worldwide crop pest, Drosophila suzukii We demonstrate that this drive system, based on an engineered maternal "toxin" coupled with a linked embryonic "antidote," is capable of biasing Mendelian inheritance rates with up to 100% efficiency. However, we find that drive resistance, resulting from naturally occurring genetic variation and associated fitness costs, can be selected for and hinder the spread of such a drive. Despite this, our results suggest that this gene drive could maintain itself at high frequencies in a wild population and spread to fixation if either its fitness costs or toxin resistance were reduced, providing a clear path forward for developing future such systems in this pest.
Gene drivesDrosophilaGene drive
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ACS chemical biology · 2018-Feb-16 · PMID 29370514
John M Marshall, Omar S Akbari
The recent discovery of CRISPR and its application as a gene editing tool has enabled a range of gene drive systems to be engineered with greater ease. In order for the benefits of this technology to be realized, in some circumstances drive systems should be developed that are capable of both spreading into populations to achieve their desired impact and being recalled in the event of unwanted consequences or public disfavor. We review the performance of three broad categories of drive systems at achieving these goals: threshold-dependent drives, homing-based drive and remediation systems, and temporally self-limiting systems such as daisy-chain drives.
Gene drivesCRISPRGene drive
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Current biology : CB · 2018-Feb-05 · PMID 29395917
Clément Vinauger, Chloé Lahondère, Gabriella H Wolff, Lauren T Locke, Jessica E Liaw, Jay Z Parrish, Omar S Akbari, Michael H Dickinson, Jeffrey A Riffell
How mosquitoes determine which individuals to bite has important epidemiological consequences. This choice is not random; most mosquitoes specialize in one or a few vertebrate host species, and some individuals in a host population are preferred over others. Mosquitoes will also blood feed from other hosts when their preferred is no longer abundant, but the mechanisms mediating these shifts between hosts, and preferences for certain individuals within a host species, remain unclear. Here, we show that olfactory learning may contribute to Aedes aegypti mosquito biting preferences and host shifts. Training and testing to scents of humans and other host species showed that mosquitoes can aversively learn the scent of specific humans and single odorants and learn to avoid the scent of rats (but not chickens). Using pharmacological interventions, RNAi, and CRISPR gene editing, we found that modification of the dopamine-1 receptor suppressed their learning abilities. We further show through combined electrophysiological and behavioral recordings from tethered flying mosquitoes that these odors evoke changes in both behavior and antennal lobe (AL) neuronal responses and that dopamine strongly modulates odor-evoked responses in AL neurons. Not only do these results provide direct experimental evidence that olfactory learning in mosquitoes can play an epidemiological role, but collectively, they also provide neuroanatomical and functional demonstration of the role of dopamine in mediating this learning-induced plasticity, for the first time in a disease vector insect.
Mosquito neurobiologyAedes aegyptiMosquitoesCRISPRRNA targeting
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G3 (Bethesda, Md.) · 2018-Feb-02 · PMID 29233915
Ming Li, Omar S Akbari, Bradley J White
Anopheles mosquitoes transmit at least 200 million annual malaria infections worldwide. Despite considerable genomic resources, mechanistic understanding of biological processes in Anopheles has been hampered by a lack of tools for reverse genetics. Here, we report successful application of the CRISPR/Cas9 system for highly efficient, site-specific mutagenesis in the diverse malaria vectors Anopheles albimanus , A. coluzzii , and A. funestus When guide RNAs (gRNAs) and Cas9 protein are injected at high concentration, germline mutations are common and usually biallelic, allowing for the rapid creation of stable mutant lines for reverse genetic analysis. Our protocol should enable researchers to dissect the molecular and cellular basis of anopheline traits critical to successful disease transmission, potentially exposing new targets for malaria control.
AnophelesMosquitoesCRISPR
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Current opinion in insect science · 2018-Aug-22 · PMID 30551769
Valentino M Gantz, Omar S Akbari
Initially discovered in bacteria, CRISPR-based genome editing endonucleases have proven remarkably amenable for adaptation to insects. To date, these endonucleases have been utilized in a plethora of both model and non-model insects including diverse flies, bees, beetles, butterflies, moths, and grasshoppers, to name a few, thereby revolutionizing functional genomics of insects. In addition to basic genome editing, they have also been invaluable for advanced genome engineering and synthetic biology applications. Here we explore the recent genome editing advancements in insects for generating site-specific genomic mutations, insertions, deletions, as well as more advanced applications such as Homology Assisted Genome Knock-in (HACK), potential to utilize DNA base editing, generating predictable reciprocal chromosomal translocations, and development gene drives to control the fate of wild populations.
Gene drivesSynthetic biologyCRISPRGene drive
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Scientific reports · 2017-Jun-19 · PMID 28630470
John M Marshall, Anna Buchman, Héctor M Sánchez C, Omar S Akbari
The recent development of a CRISPR-Cas9-based homing system for the suppression of Anopheles gambiae is encouraging; however, with current designs, the slow emergence of homing-resistant alleles is expected to result in suppressed populations rapidly rebounding, as homing-resistant alleles have a significant fitness advantage over functional, population-suppressing homing alleles. To explore this concern, we develop a mathematical model to estimate tolerable rates of homing-resistant allele generation to suppress a wild population of a given size. Our results suggest that, to achieve meaningful population suppression, tolerable rates of resistance allele generation are orders of magnitude smaller than those observed for current designs for CRISPR-Cas9-based homing systems. To remedy this, we theoretically explore a homing system architecture in which guide RNAs (gRNAs) are multiplexed, increasing the effective homing rate and decreasing the effective resistant allele generation rate. Modeling results suggest that the size of the population that can be suppressed increases exponentially with the number of multiplexed gRNAs and that, with four multiplexed gRNAs, a mosquito species could potentially be suppressed on a continental scale. We also demonstrate successful proof-of-principle use of multiplexed ribozyme flanked gRNAs to induce mutations in vivo in Drosophila melanogaster - a strategy that could readily be adapted to engineer stable, homing-based drives in relevant organisms.
Gene drivesVector controlAnophelesDrosophilaMosquitoesCRISPR
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Journal of visualized experiments : JoVE · 2017-Dec-25 · PMID 29364231
Ming Li, Michelle Bui, Omar S Akbari
The jewel wasp Nasonia vitripennis has emerged as an effective model system for the study of processes including sex determination, haplo-diploid sex determination, venom synthesis, and host-symbiont interactions, among others. A major limitation of working with this organism is the lack of effective protocols to perform directed genome modifications. An important part of genome modification is delivery of editing reagents, including CRISPR/Cas9 molecules, into embryos through microinjection. While microinjection is well established in many model organisms, this technique is particularly challenging to perform in N. vitripennis primarily due to its small embryo size, and the fact that embryonic development occurs entirely within a parasitized blowfly pupa. The following procedure overcomes these significant challenges while demonstrating a streamlined, visual procedure for effectively removing wasp embryos from parasitized host pupae, microinjecting them, and carefully transplanting them back into the host for continuation and completion of development. This protocol will strongly enhance the capability of research groups to perform advanced genome modifications in this organism.
Sex determinationCRISPR
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Pathogens and global health · 2017-Dec-23 · PMID 29566584
Robyn Raban, Omar S Akbari
No abstract is available from PubMed.
Gene drivesGene drive
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Chromosoma · 2017-Dec-20 · PMID 28821911
Yue Li, Xueyuan A Jing, John C Aldrich, C Clifford, Jian Chen, Omar S Akbari, Patrick M Ferree
No abstract is available from PubMed.
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Proceedings of the National Academy of Sciences of the United States of America · 2017-Dec-05 · PMID 29138316
Ming Li, Michelle Bui, Ting Yang, Christian S Bowman, Bradley J White, Omar S Akbari
The development of CRISPR/Cas9 technologies has dramatically increased the accessibility and efficiency of genome editing in many organisms. In general, in vivo germline expression of Cas9 results in substantially higher activity than embryonic injection. However, no transgenic lines expressing Cas9 have been developed for the major mosquito disease vector Aedes aegypti Here, we describe the generation of multiple stable, transgenic Ae. aegypti strains expressing Cas9 in the germline, resulting in dramatic improvements in both the consistency and efficiency of genome modifications using CRISPR. Using these strains, we disrupted numerous genes important for normal morphological development, and even generated triple mutants from a single injection. We have also managed to increase the rates of homology-directed repair by more than an order of magnitude. Given the exceptional mutagenic efficiency and specificity of the Cas9 strains we engineered, they can be used for high-throughput reverse genetic screens to help functionally annotate the Ae. aegypti genome. Additionally, these strains represent a step toward the development of novel population control technologies targeting Ae. aegypti that rely on Cas9-based gene drives.
Gene drivesVector controlSynthetic biologyAedes aegyptiMosquitoesCRISPR
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Chromosoma · 2017-Dec-05 · PMID 28780664
Yue Li, Xueyuan A Jing, John C Aldrich, C Clifford, Jian Chen, Omar S Akbari, Patrick M Ferree
B chromosomes are found in numerous plants and animals. These nonessential, supernumerary chromosomes are often composed primarily of noncoding DNA repeats similar to those found within transcriptionally "silenced" heterochromatin. In order to persist within their resident genomes, many B chromosomes exhibit exceptional cellular behaviors, including asymmetric segregation into gametes and induction of genome elimination during early development. An important goal in understanding these behaviors is to identify unique B chromosome sequences and characterize their transcriptional contributions. We investigated these properties by examining a paternally transmitted B chromosome known as paternal sex ratio (PSR), which is present in natural populations of the jewel wasp Nasonia vitripennis. To facilitate its own transmission, PSR severely biases the sex ratio by disrupting early chromatin remodeling processes. Through cytological mapping and other approaches, we identified multiple DNA repeats unique to PSR, as well as those found on the A chromosomes, suggesting that PSR arose through a merger of sequences from both within and outside the N. vitripennis genome. The majority of PSR-specific repeats are interspersed among each other across PSR's long arm, in contrast with the distinct "blocks" observed in other organisms' heterochromatin. Through transcriptional profiling, we identified a subset of repeat-associated, small RNAs expressed by PSR, most of which map to a single PSR-specific repeat. These RNAs are expressed at much higher levels than those arising from A chromosome-linked repeats, suggesting that in addition to its sequence organization, PSR's transcriptional properties differ substantially from the pericentromeric regions of the normal chromosomes.
Sex determination
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Nature biotechnology · 2017-Aug-08 · PMID 28787415
Zach Adelman, Omar Akbari, John Bauer, Ethan Bier, Cinnamon Bloss, Sarah R Carter, Craig Callender, Adriana Costero-Saint Denis, Peter Cowhey, Brinda Dass, Jason Delborne, Mary Devereaux, Peter Ellsworth, Robert M Friedman, Valentino Gantz, Clark Gibson, Bruce A Hay, Mark Hoddle, Anthony A James, Stephanie James, Lyric Jorgenson, Michael Kalichman, John Marshall, William McGinnis, Jack Newman, Alan Pearson, Hector Quemada, Larisa Rudenko, Anthony Shelton, Joseph M Vinetz, Jennifer Weisman, Brenda Wong, Chris Wozniak
No abstract is available from PubMed.
Gene drivesGene drive
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Scientific reports · 2017-Apr-19 · PMID 28424460
Ming Li, Lauren Yun Cook Au, Deema Douglah, Abigail Chong, Bradley J White, Patrick M Ferree, Omar S Akbari
The revolutionary RNA-guided endonuclease CRISPR/Cas9 system has proven to be a powerful tool for gene editing in a plethora of organisms. Here, utilizing this system we developed an efficient protocol for the generation of heritable germline mutations in the parasitoid jewel wasp, Nasonia vitripennis, a rising insect model organism for the study of evolution, development of axis pattern formation, venom production, haplo-diploid sex determination, and host-symbiont interactions. To establish CRISPR-directed gene editing in N. vitripennis, we targeted a conserved eye pigmentation gene cinnabar, generating several independent heritable germline mutations in this gene. Briefly, to generate these mutants, we developed a protocol to efficiently collect N. vitripennis eggs from a parasitized flesh fly pupa, Sarcophaga bullata, inject these eggs with Cas9/guide RNA mixtures, and transfer injected eggs back into the host to continue development. We also describe a flow for screening mutants and establishing stable mutant strains through genetic crosses. Overall, our results demonstrate that the CRISPR/Cas9 system is a powerful tool for genome manipulation in N. vitripennis, with strong potential for expansion to target critical genes, thus allowing for the investigation of several important biological phenomena in this organism.
Sex determinationCRISPR
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Development (Cambridge, England) · 2016-Oct-01 · PMID 27702788
Harry M T Choi, Colby R Calvert, Naeem Husain, David Huss, Julius C Barsi, Benjamin E Deverman, Ryan C Hunter, Mihoko Kato, S Melanie Lee, Anna C T Abelin, Adam Z Rosenthal, Omar S Akbari, Yuwei Li, Bruce A Hay, Paul W Sternberg, Paul H Patterson, Eric H Davidson, Sarkis K Mazmanian, David A Prober, Matt van de Rijn, Jared R Leadbetter, Dianne K Newman, Carol Readhead, Marianne E Bronner, Barbara Wold, Rusty Lansford, Tatjana Sauka-Spengler, Scott E Fraser, Niles A Pierce
In situ hybridization methods are used across the biological sciences to map mRNA expression within intact specimens. Multiplexed experiments, in which multiple target mRNAs are mapped in a single sample, are essential for studying regulatory interactions, but remain cumbersome in most model organisms. Programmable in situ amplifiers based on the mechanism of hybridization chain reaction (HCR) overcome this longstanding challenge by operating independently within a sample, enabling multiplexed experiments to be performed with an experimental timeline independent of the number of target mRNAs. To assist biologists working across a broad spectrum of organisms, we demonstrate multiplexed in situ HCR in diverse imaging settings: bacteria, whole-mount nematode larvae, whole-mount fruit fly embryos, whole-mount sea urchin embryos, whole-mount zebrafish larvae, whole-mount chicken embryos, whole-mount mouse embryos and formalin-fixed paraffin-embedded human tissue sections. In addition to straightforward multiplexing, in situ HCR enables deep sample penetration, high contrast and subcellular resolution, providing an incisive tool for the study of interlaced and overlapping expression patterns, with implications for research communities across the biological sciences.
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Current biology : CB · 2016-May-23 · PMID 27161498
Becky Cheng, Nitin Kuppanda, John C Aldrich, Omar S Akbari, Patrick M Ferree
Numerous arthropods harbor maternally transmitted bacteria that induce the preferential death of males [1-7]. This sex-specific lethality benefits the bacteria because males are "dead ends" regarding bacterial transmission, and their absence may result in additional resources for their viable female siblings who can thereby more successfully transmit the bacteria [5]. Although these symbionts disrupt a range of developmental processes [8-10], the underlying cellular mechanisms are largely unknown. It was previously shown that mutations in genes of the dosage compensation pathway of Drosophila melanogaster suppressed male killing caused by the bacterium, Spiroplasma [10]. This result suggested that dosage compensation is a target of Spiroplasma. However, it remains unclear how this pathway is affected, and whether the underlying interactions require the male-specific cellular environment. Here, we investigated the cellular basis of male embryonic lethality in D. melanogaster induced by Spiroplasma. We found that the dosage compensation complex (DCC), which acetylates X chromatin in males [11], becomes mis-localized to ectopic regions of the nucleus immediately prior to the killing phase. This effect was accompanied by inappropriate histone acetylation and genome-wide mis-regulation of gene expression. Artificially induced formation of the DCC in infected females, through transgenic expression of the DCC-specific gene msl-2, resulted in mis-localization of this complex to non-X regions and early Spiroplasma-induced death, mirroring the killing effects in males. These findings strongly suggest that Spiroplasma initiates male killing by targeting the dosage compensation machinery directly and independently of other cellular features characteristic of the male sex.
Synthetic biologyDrosophilaTransgenesis
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Nature reviews. Genetics · 2016-Mar-15 · PMID 26875679
Jackson Champer, Anna Buchman, Omar S Akbari
Engineered gene drives - the process of stimulating the biased inheritance of specific genes - have the potential to enable the spread of desirable genes throughout wild populations or to suppress harmful species, and may be particularly useful for the control of vector-borne diseases such as malaria. Although several types of selfish genetic elements exist in nature, few have been successfully engineered in the laboratory thus far. With the discovery of RNA-guided CRISPR-Cas9 (clustered regularly interspaced short palindromic repeats-CRISPR-associated 9) nucleases, which can be utilized to create, streamline and improve synthetic gene drives, this is rapidly changing. Here, we discuss the different types of engineered gene drives and their potential applications, as well as current policies regarding the safety and regulation of gene drives for the manipulation of wild populations.
Gene drivesCRISPRGene drive
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Proceedings of the National Academy of Sciences of the United States of America · 2016-Apr-12 · PMID 27035980
Andrew Brantley Hall, Philippos-Aris Papathanos, Atashi Sharma, Changde Cheng, Omar S Akbari, Lauren Assour, Nicholas H Bergman, Alessia Cagnetti, Andrea Crisanti, Tania Dottorini, Elisa Fiorentini, Roberto Galizi, Jonathan Hnath, Xiaofang Jiang, Sergey Koren, Tony Nolan, Diane Radune, Maria V Sharakhova, Aaron Steele, Vladimir A Timoshevskiy, Nikolai Windbichler, Simo Zhang, Matthew W Hahn, Adam M Phillippy, Scott J Emrich, Igor V Sharakhov, Zhijian Jake Tu, Nora J Besansky
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.
Sex determinationAnophelesMosquitoes
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G3 (Bethesda, Md.) · 2015-Oct-13 · PMID 26464360
Patrick M Ferree, Christopher Fang, Mariah Mastrodimos, Bruce A Hay, Henry Amrhein, Omar S Akbari
The jewel wasp Nasonia vitripennis is a rising model organism for the study of haplo-diploid reproduction characteristic of hymenopteran insects, which include all wasps, bees, and ants. We performed transcriptional profiling of the ovary, the female soma, and the male soma of N. vitripennis to complement a previously existing transcriptome of the wasp testis. These data were deposited into an open-access genome browser for visualization of transcripts relative to their gene models. We used these data to identify the assemblies of genes uniquely expressed in the germ-line tissues. We found that 156 protein-coding genes are expressed exclusively in the wasp testis compared with only 22 in the ovary. Of the testis-specific genes, eight are candidates for male-specific DNA packaging proteins known as protamines. We found very similar expression patterns of centrosome associated genes in the testis and ovary, arguing that de novo centrosome formation, a key process for development of unfertilized eggs into males, likely does not rely on large-scale transcriptional differences between these tissues. In contrast, a number of meiosis-related genes show a bias toward testis-specific expression, despite the lack of true meiosis in N. vitripennis males. These patterns may reflect an unexpected complexity of male gamete production in the haploid males of this organism. Broadly, these data add to the growing number of genomic and genetic tools available in N. vitripennis for addressing important biological questions in this rising insect model organism.
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Current biology : CB · 2015-Oct-05 · PMID 26439332
Juan Li, Alejandra I Olvera, Omar S Akbari, Annie Moradian, Michael J Sweredoski, Sonja Hess, Bruce A Hay
Development of non-surgical methods of long-term or permanent contraception remains a challenge. Towards this objective, we show that intramuscular injection of a replication-incompetent, recombinant adeno-associated virus (rAAV) designed to express an antibody that binds gonadotropin-releasing hormone (GnRH), a master regulator of reproduction in vertebrates, results in long-term infertility in male and female mice. Female mice are also rendered infertile through rAAV-dependent expression of an antibody that binds to the zona pellucida (ZP), a glycoprotein matrix that surrounds the egg and functions as a sperm-binding site. Many proteins known or suspected to be important for reproduction can be targeted, potentially reversibly, using this approach, which we refer to as vectored contraception (VC).
Antiviral engineering
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Science (New York, N.Y.) · 2015-Aug-28 · PMID 26229113
Omar S Akbari, Hugo J Bellen, Ethan Bier, Simon L Bullock, Austin Burt, George M Church, Kevin R Cook, Peter Duchek, Owain R Edwards, Kevin M Esvelt, Valentino M Gantz, Kent G Golic, Scott J Gratz, Melissa M Harrison, Keith R Hayes, Anthony A James, Thomas C Kaufman, Juergen Knoblich, Harmit S Malik, Kathy A Matthews, Kate M O'Connor-Giles, Annette L Parks, Norbert Perrimon, Fillip Port, Steven Russell, Ryu Ueda, Jill Wildonger
No abstract is available from PubMed.
Gene drivesGene drive
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Scientific reports · 2014-Feb-04 · PMID 24492376
Omar S Akbari, Philippos A Papathanos, Jeremy E Sandler, Katie Kennedy, Bruce A Hay
The mosquito Aedes aegypti is the principal vector for the yellow fever and dengue viruses, and is also responsible for recent outbreaks of the alphavirus chikungunya. Vector control strategies utilizing engineered gene drive systems are being developed as a means of replacing wild, pathogen transmitting mosquitoes with individuals refractory to disease transmission, or bringing about population suppression. Several of these systems, including Medea, UD(MEL), and site-specific nucleases, which can be used to drive genes into populations or bring about population suppression, utilize transcriptional regulatory elements that drive germline-specific expression. Here we report the identification of multiple regulatory elements able to drive gene expression specifically in the female germline, or in the male and female germline, in the mosquito Aedes aegypti. These elements can also be used as tools with which to probe the roles of specific genes in germline function and in the early embryo, through overexpression or RNA interference.
Gene drivesAntiviral engineeringVector controlAedes aegyptiMosquitoesGene drive
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ACS synthetic biology · 2014-Dec-19 · PMID 23654248
Omar S Akbari, Chun-Hong Chen, John M Marshall, Haixia Huang, Igor Antoshechkin, Bruce A Hay
Insects act as vectors for diseases of plants, animals, and humans. Replacement of wild insect populations with genetically modified individuals unable to transmit disease provides a potentially self-perpetuating method of disease prevention. Population replacement requires a gene drive mechanism in order to spread linked genes mediating disease refractoriness through wild populations. We previously reported the creation of synthetic Medea selfish genetic elements able to drive population replacement in Drosophila. These elements use microRNA-mediated silencing of myd88, a maternally expressed gene required for embryonic dorso-ventral pattern formation, coupled with early zygotic expression of a rescuing transgene, to bring about gene drive. Medea elements that work through additional mechanisms are needed in order to be able to carry out cycles of population replacement and/or remove existing transgenes from the population, using second-generation elements that spread while driving first-generation elements out of the population. Here we report the synthesis and population genetic behavior of two new synthetic Medea elements that drive population replacement through manipulation of signaling pathways involved in cellular blastoderm formation or Notch signaling, demonstrating that in Drosophila Medea elements can be generated through manipulation of diverse signaling pathways. We also describe the mRNA and small RNA changes in ovaries and early embryos associated from Medea-bearing females. Finally, we use modeling to illustrate how Medea elements carrying genes that result in diapause-dependent female lethality could be used to bring about population suppression.
Gene drivesVector controlDrosophilaGene drive
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G3 (Bethesda, Md.) · 2013-Sep-04 · PMID 23833213
Omar S Akbari, Igor Antoshechkin, Henry Amrhein, Brian Williams, Race Diloreto, Jeremy Sandler, Bruce A Hay
Mosquitoes are vectors of a number of important human and animal diseases. The development of novel vector control strategies requires a thorough understanding of mosquito biology. To facilitate this, we used RNA-seq to identify novel genes and provide the first high-resolution view of the transcriptome throughout development and in response to blood feeding in a mosquito vector of human disease, Aedes aegypti, the primary vector for Dengue and yellow fever. We characterized mRNA expression at 34 distinct time points throughout Aedes development, including adult somatic and germline tissues, by using polyA+ RNA-seq. We identify a total of 14,238 novel new transcribed regions corresponding to 12,597 new loci, as well as many novel transcript isoforms of previously annotated genes. Altogether these results increase the annotated fraction of the transcribed genome into long polyA+ RNAs by more than twofold. We also identified a number of patterns of shared gene expression, as well as genes and/or exons expressed sex-specifically or sex-differentially. Expression profiles of small RNAs in ovaries, early embryos, testes, and adult male and female somatic tissues also were determined, resulting in the identification of 38 new Aedes-specific miRNAs, and ~291,000 small RNA new transcribed regions, many of which are likely to be endogenous small-interfering RNAs and Piwi-interacting RNAs. Genes of potential interest for transgene-based vector control strategies also are highlighted. Our data have been incorporated into a user-friendly genome browser located at www.Aedes.caltech.edu, with relevant links to Vectorbase (www.vectorbase.org).
Antiviral engineeringVector controlAedes aegyptiMosquitoes
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G3 (Bethesda, Md.) · 2013-Sep-04 · PMID 23893741
Omar S Akbari, Igor Antoshechkin, Bruce A Hay, Patrick M Ferree
A widespread phenomenon in nature is sex ratio distortion of arthropod populations caused by microbial and genetic parasites. Currently little is known about how these agents alter host developmental processes to favor one sex or the other. The paternal sex ratio (PSR) chromosome is a nonessential, paternally transmitted centric fragment that segregates in natural populations of the jewel wasp, Nasonia vitripennis. To persist, PSR is thought to modify the hereditary material of the developing sperm, with the result that all nuclear DNA other than the PSR chromosome is destroyed shortly after fertilization. This results in the conversion of a fertilized embryo--normally a female--into a male, thereby insuring transmission of the "selfish" PSR chromosome, and simultaneously leading to wasp populations that are male-biased. To begin to understand this system at the mechanistic level, we carried out transcriptional profiling of testis from WT and PSR-carrying males. We identified a number of transcripts that are differentially expressed between these conditions. We also discovered nine transcripts that are uniquely expressed from the PSR chromosome. Four of these PSR-specific transcripts encode putative proteins, whereas the others have very short open reading frames and no homology to known proteins, suggesting that they are long noncoding RNAs. We propose several different models for how these transcripts could facilitate PSR-dependent effects. Our analyses also revealed 15.71 MB of novel transcribed regions in the N. vitripennis genome, thus increasing the current annotation of total transcribed regions by 53.4%. Finally, we detected expression of multiple meiosis-related genes in the wasp testis, despite the lack of conventional meiosis in the male sex.
Sex determination
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Current biology : CB · 2013-Apr-22 · PMID 23541732
Omar S Akbari, Kelly D Matzen, John M Marshall, Haixia Huang, Catherine M Ward, Bruce A Hay
Replacement of wild insect populations with genetically modified individuals unable to transmit disease provides a self-perpetuating method of disease prevention but requires a gene drive mechanism to spread these traits to high frequency. Drive mechanisms requiring that transgenes exceed a threshold frequency in order to spread are attractive because they bring about local but not global replacement, and transgenes can be eliminated through dilution of the population with wild-type individuals. These features are likely to be important in many social and regulatory contexts. Here we describe the first creation of a synthetic threshold-dependent gene drive system, designated maternal-effect lethal underdominance (UD(MEL)), in which two maternally expressed toxins, located on separate chromosomes, are each linked with a zygotic antidote able to rescue maternal-effect lethality of the other toxin. We demonstrate threshold-dependent replacement in single- and two-locus configurations in Drosophila. Models suggest that transgene spread can often be limited to local environments. They also show that in a population in which single-locus UD(MEL) has been carried out, repeated release of wild-type males can result in population suppression, a novel method of genetic population manipulation.
Gene drivesVector controlDrosophilaGene drive
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PloS one · 2011-Jan-21 · PMID 21283702
Margaret C W Ho, Benjamin J Schiller, Omar S Akbari, Esther Bae, Robert A Drewell
There are many examples within gene complexes of transcriptional enhancers interacting with only a subset of target promoters. A number of molecular mechanisms including promoter competition, insulators and chromatin looping are thought to play a role in regulating these interactions. At the Drosophila bithorax complex (BX-C), the IAB5 enhancer specifically drives gene expression only from the Abdominal-B (Abd-B) promoter, even though the enhancer and promoter are 55 kb apart and are separated by at least three insulators. In previous studies, we discovered that a 255 bp cis-regulatory module, the promoter tethering element (PTE), located 5' of the Abd-B transcriptional start site is able to tether IAB5 to the Abd-B promoter in transgenic embryo assays. In this study we examine the functional role of the PTE at the endogenous BX-C using transposon-mediated mutagenesis. Disruption of the PTE by P element insertion results in a loss of enhancer-directed Abd-B expression during embryonic development and a homeotic transformation of abdominal segments. A partial deletion of the PTE and neighboring upstream genomic sequences by imprecise excision of the P element also results in a similar loss of Abd-B expression in embryos. These results demonstrate that the PTE is an essential component of the regulatory network at the BX-C and is required in vivo to mediate specific long-range enhancer-promoter interactions.
Synthetic biologyDrosophilaTransgenesis
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BMC cell biology · 2010-Dec-31 · PMID 21194420
Daniel Oliver, Brian Sheehan, Heather South, Omar Akbari, Chi-Yun Pai
Chromatin insulators or boundary elements are a class of functional elements in the eukaryotic genome. They regulate gene transcription by interfering with promoter-enhancer communication. The Cp190 protein of Drosophila melanogaster is essential to the function of at least three-types of chromatin insulator complexes organized by Su(Hw), CTCF and BEAF32.
We mapped functional regions of Cp190 in vivo and identified three domains that are essential for the insulator function and for the viability of flies: the BTB/POZ domain, an aspartic acid-rich (D-rich) region and a C-terminal glutamic acid-rich (E-rich) region. Other domains including the centrosomal targeting domain and the zinc fingers are dispensable. The N-terminal CP190BTB-D fragment containing the BTB/POZ domain and the D-rich region is sufficient to mediate association with all three types of insulator complexes. The fragment however is not sufficient for insulator activity or viability. The Cp190 and CP190BTB-D are regulated differently in cells treated with heat-shock. The Cp190 dissociated from chromosomes during heat-shock, indicating that dissociation of Cp190 with chromosomes can be regulated. In contrast, the CP190BTB-D fragment didn't dissociate from chromosomes in the same heat-shocked condition, suggesting that the deleted C-terminal regions have a role in regulating the dissociation of Cp190 with chromosomes.
The N-terminal fragment of Cp190 containing the BTB/POZ domain and the D-rich region mediates association of Cp190 with all three types of insulator complexes and that the E-rich region of Cp190 is required for dissociation of Cp190 from chromosomes during heat-shock. The heat-shock-induced dissociation is strong evidence indicating that dissociation of the essential insulator protein Cp190 from chromosomes is regulated. Our results provide a mechanism through which activities of an insulator can be modulated by internal and external cues.
Drosophila
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BMC cell biology · 2009-Jan-29 · PMID 19178707
Omar S Akbari, Daniel Oliver, Katie Eyer, Chi-Yun Pai
Tagged fusion proteins are priceless tools for monitoring the activities of biomolecules in living cells. However, over-expression of fusion proteins sometimes leads to the unwanted lethality or developmental defects. Therefore, vectors that can express tagged proteins at physiological levels are desirable tools for studying dosage-sensitive proteins. We developed a set of Entry/Gateway vectors for expressing fluorescent fusion proteins in Drosophila melanogaster. The vectors were used to generate fluorescent CP190 which is a component of the gypsy chromatin insulator. We used the fluorescent CP190 to study the dynamic movement of related chromatin insulators in living cells.
The Entry/Gateway system is a timesaving technique for quickly generating expression constructs of tagged fusion proteins. We described in this study an Entry/Gateway based system, which includes six P-element destination vectors (P-DEST) for expressing tagged proteins (eGFP, mRFP, or myc) in Drosophila melanogaster and a TA-based cloning vector for generating entry clones from unstable DNA sequences. We used the P-DEST vectors to express fluorecent CP190 at tolerable levels. Expression of CP190 using the UAS/Gal4 system, instead, led to either lethality or underdeveloped tissues. The expressed eGFP- or mRFP-tagged CP190 proteins are fully functional and rescued the lethality of the homozygous CP190 mutation. We visualized a wide range of CP190 distribution patterns in living cell nuclei, from thousands of tiny particles to less than ten giant ones, which likely reflects diverse organization of higher-order chromatin structures. We also visualized the fusion of multiple smaller insulator bodies into larger aggregates in living cells, which is likely reflective of the dynamic activities of reorganization of chromatin in living nuclei.
We have developed an efficient cloning system for expressing dosage-sensitive proteins in Drosophila melanogaster. This system successfully expresses functional fluorescent CP190 fusion proteins. The fluorescent CP190 proteins exist in insulator bodies of various numbers and sizes among cells from multiple living tissues. Furthermore, live imaging of the movements of these fluorescent-tagged proteins suggests that the assembly and disassembly of insulator bodies are normal activities in living cells and may be directed for regulating transcription.
Drosophila
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Development (Cambridge, England) · 2008-Jan-28 · PMID 18045839
Omar S Akbari, Esther Bae, Holly Johnsen, Alfred Villaluz, Debbie Wong, Robert A Drewell
A key question in our understanding of the cis-regulation of gene expression during embryonic development has been the molecular mechanism that directs enhancers to specific promoters within a gene complex. Promoter competition and insulators are thought to play a role in regulating these interactions. In the bithorax complex of Drosophila, the IAB5 enhancer is located 55 kb 3' of the Abdominal-B (Abd-B) promoter and 48 kb 5' of the abdominal-A (abd-A) promoter. Although roughly equidistant from the two promoters, IAB5 specifically interacts only with the Abdominal-B promoter, even though the enhancer and promoter are separated by at least two insulators. Here we demonstrate that a 255 bp element, located 40 bp 5' of the Abd-B transcriptional start site, has a novel cis-regulatory activity as it is able to tether IAB5 to the Abd-B promoter in transgenic embryos. The tethering element is sufficient to direct IAB5 to an ectopic promoter in competition assays. Deletion of the promoter-tethering element results in the redirection of enhancer-driven gene expression on transgenes. Taken together, these results provide evidence that specific long-range enhancer-promoter interactions in the bithorax complex are regulated by a tethering element 5' of the Abd-B promoter. We discuss a bioinformatic analysis of the tethering element across different Drosophila species and a possible molecular mechanism by which this element functions. We also examine existing evidence that this novel class of cis-regulatory elements might regulate enhancer-promoter specificity at other gene complexes.
Synthetic biologyDrosophilaTransgenesis
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Fly · 2007-5-28 · PMID 18504507
Omar S Akbari, Ben J Schiller, Sara E Goetz, Margaret C W Ho, Esther Bae, Robert A Drewell
At the Drosophila bithorax complex many distinct classes of cis-regulatory modules work collectively during development to control gene expression. Abdominal-B (Abd-B) is one of three homeotic genes in the BX-C and is expressed in specific presumptive abdominal segments in the embryo. The transcription of Abd-B is tightly controlled by an array of cis-regulatory modules that direct its expression over extended genomic distances. These regulatory modules include promoters, insulators, silencers, enhancers, promoter targeting sequences and the recently identified promoter tethering element (PTE). To activate gene expression at the endogenous complex, enhancers located >50 kb away must bypass intervening insulators to interact with the Abd-B promoter. The molecular mechanisms that allow enhancers to bypass insulators are not currently well understood. In this short article, we report on a novel mechanism for insulator bypass involving the PTE. In addition, we use bioinformatic analysis across twelve Drosophila genomes to identify putative cis-regulatory sequences that may be capable of facilitating specific promoter-enhancer interactions at the bithorax complex and propose a model for their molecular function during development.
Drosophila
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Developmental biology · 2006-May-15 · PMID 16545794
Omar S Akbari, Adam Bousum, Esther Bae, Robert A Drewell
Genome sequencing has revealed that in metazoans, only a small percentage of DNA actually codes for functional proteins. Research efforts have focused on elucidating the purpose of the rest of the genome, which was initially largely thought of as mere 'junk' DNA. One genomic region that is proving to be a rich source of new information is the Drosophila bithorax complex (BX-C). At this homeotic gene complex, many different classes of cis-regulatory elements, such as insulators, silencers, enhancers, and promoters, work together to tightly control gene expression during development. Recent studies have begun to unravel the intricate nature of these regulatory interactions. The BX-C was first discovered and characterized by Ed Lewis over three decades ago. In his seminal 1978 Nature paper, Lewis speculated that "substances" originating from the nongenic regions of the BX-C may regulate expression of the neighboring abdominal-A and Abdominal-B homeotic genes. A number of discoveries in the last few years suggest that he was right. The activation of some of the cis-sequences at the complex appears to be controlled by nongenic transcription, providing a further level of regulatory complexity to regions of nonprotein coding DNA. The hope is that these studies of gene regulation at the BX-C in the humble fruit fly will provide clues as to how vast intergenic regions contribute to the incredible complexity of gene regulation in other species, including humans.
Drosophila
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