scholarly journals Germline Cas9 Expression Yields Highly Efficient Genome Engineering in a Major Worldwide Disease Vector, Aedes aegypti

2017 ◽  
Author(s):  
Ming Li ◽  
Michelle Bui ◽  
Ting Yang ◽  
Bradley J. White ◽  
Omar S. Akbari
Keyword(s):  
Aedes Aegypti ◽  
High Throughput ◽  
Disease Transmission ◽  
Genome Engineering ◽  
Population Control ◽  
Control Strategies ◽  
Morphological Development ◽  
Disease Vector ◽  
Genome Modifications ◽  
Gene Drives

AbstractThe 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 built, they can be used for high-throughput reverse genetic screens to help functionally annotate the Ae. aegypti genome. Additionally, these strains represent a first step towards the development of novel population control technologies targeting Ae. aegypti that rely on Cas9-based gene drives.Significance StatementAedes aegypti is the principal vector of multiple arboviruses that significantly affect human health including dengue, chikungunya, and zika. Development of tools for efficient genome engineering in this mosquito will not only lay the foundation for the application of novel genetic control strategies that do not rely on insecticides, but will also accelerate basic research on key biological processes involved in disease transmission. Here, we report the development of a transgenic CRISPR approach for rapid gene disruption in this organism. Given their high editing efficiencies, the Cas9 strains we developed can be used to quickly generate novel genome modifications allowing for high-throughput gene targeting, and can possibly facilitate the development of gene drives, thereby accelerating comprehensive functional annotation and development of innovative population control strategies for Ae. aegypti.

scholarly journals Germline Cas9 expression yields highly efficient genome engineering in a major worldwide disease vector, Aedes aegypti

2017 ◽  
Vol 114 (49) ◽  
pp. E10540-E10549 ◽  
Author(s):  
Ming Li ◽  
Michelle Bui ◽  
Ting Yang ◽  
Christian S. Bowman ◽  
Bradley J. White ◽  
...  
Keyword(s):  
Aedes Aegypti ◽  
Genome Engineering ◽  
Population Control ◽  
Morphological Development ◽  
Disease Vector ◽  
Transgenic Lines ◽  
Germline Expression ◽  
Order Of Magnitude ◽  
Control Technologies

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.

The Global Expansion of Dengue: How Aedes aegypti Mosquitoes Enabled the First Pandemic Arbovirus

2020 ◽  
Vol 65 (1) ◽  
pp. 191-208 ◽  
Author(s):  
Oliver J. Brady ◽  
Simon I. Hay
Keyword(s):  
Invasive Species ◽  
Aedes Aegypti ◽  
Infectious Diseases ◽  
Control Strategies ◽  
Viral Disease ◽  
Rapid Rise ◽  
Disease Vector ◽  
Research Priority ◽  
Global Spread ◽  
Human Habitation

Dengue is an emerging viral disease principally transmitted by the Aedes ( Stegomyia) aegypti mosquito. It is one of the fastest-growing global infectious diseases, with 100–400 million new infections a year, and is now entrenched in a growing number of tropical megacities. Behind this rapid rise is the simple adaptation of Ae. aegypti to a new entomological niche carved out by human habitation. This review describes the expansion of dengue and explores how key changes in the ecology of Ae. aegypti allowed it to become a successful invasive species and highly efficient disease vector. We argue that characterizing geographic heterogeneity in mosquito bionomics will be a key research priority that will enable us to better understand future dengue risk and design control strategies to reverse its global spread.

scholarly journals The Developmental Transcriptome of Ae. albopictus, a Major Worldwide Human Disease Vector

2019 ◽  
Author(s):  
Stephanie Gamez ◽  
Igor Antoshechkin ◽  
Stelia C. Mendez-Sanchez ◽  
Omar S. Akbari
Keyword(s):  
Aedes Aegypti ◽  
Aedes Albopictus ◽  
Human Disease ◽  
Control Strategies ◽  
Expression Profiles ◽  
Expression Patterns ◽  
Significant Differential Expression ◽  
Disease Vector ◽  
Biological Studies ◽  
Similarities And Differences

AbstractAedes albopictus mosquitoes are important vectors for a number of human pathogens including the Zika, dengue, and chikungunya viruses. Capable of displacing Aedes aegypti populations, it 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 47 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. Moreover, to illuminate the similarities and differences between Aedes aegypti, a related human disease vector, we performed a comparative analysis using the two developmental transcriptomes. We identify life stages were the two species exhibited significant differential expression among orthologs. 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 mosquitoes and provide a goldmine resource for the development of transgene-based vector control strategies.

scholarly journals Genetic incompatibility combined with female-lethality is effective and robust in simulations of Aedes aegypti population control

2018 ◽  
Author(s):  
Maciej Maselko ◽  
Stephen Heinsch ◽  
Siba Das ◽  
Michael J. Smanski
Keyword(s):  
Aedes Aegypti ◽  
Simulation Modeling ◽  
Population Control ◽  
Genetic Resistance ◽  
Effective Strategy ◽  
Agent Based Simulation ◽  
Genetic Incompatibility ◽  
Agent Based ◽  
Gene Drives ◽  
Aegypti Population

Recent reports of CRISPR/Cas9-based suppression gene drives in insects underscore the challenge of overcoming genetic resistance. Here we present results from agent-based simulation modeling of a novel Field-Amplified Male Sterility System (FAMSS) that outperforms suppression gene drives when challenged with genetic resistance. FAMSS combines a recently described synthetic genetic incompatibility approach with previously demonstrated female-lethality constructs. Our results suggest that FAMSS will be an effective strategy for temporally and spatially self-limited suppression of the disease vectoring mosquito, Aedes aegypti.

scholarly journals Assessment of a Split Homing Based Gene Drive for Efficient Knockout of Multiple Genes

2019 ◽  
Vol 10 (2) ◽  
pp. 827-837 ◽  
Author(s):  
Nikolay P. Kandul ◽  
Junru Liu ◽  
Anna Buchman ◽  
Valentino M. Gantz ◽  
Ethan Bier ◽  
...  
Keyword(s):  
Disease Transmission ◽  
Target Genes ◽  
High Efficiency ◽  
Population Control ◽  
Natural Populations ◽  
Pathogen Transmission ◽  
Gene Drive ◽  
Guide Rnas ◽  
Cas9 Protein ◽  
Gene Drives

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.

scholarly journals Assembly of the Genome of the Disease Vector Aedes aegypti onto a Genetic Linkage Map Allows Mapping of Genes Affecting Disease Transmission

2014 ◽  
Vol 8 (1) ◽  
pp. e2652 ◽  
Author(s):  
Punita Juneja ◽  
Jewelna Osei-Poku ◽  
Yung S. Ho ◽  
Cristina V. Ariani ◽  
William J. Palmer ◽  
...  
Keyword(s):  
Aedes Aegypti ◽  
Linkage Map ◽  
Disease Transmission ◽  
Genetic Linkage ◽  
Genetic Linkage Map ◽  
Disease Vector

scholarly journals Disrupting female flight in the vector Aedes aegypti

2019 ◽  
Author(s):  
Sarah O’Leary ◽  
Zach N. Adelman
Keyword(s):  
Aedes Aegypti ◽  
Flight Muscle ◽  
Population Control ◽  
Gene Knockout ◽  
Control Strategies ◽  
Specific Gene ◽  
Source Reduction ◽  
Guide Rnas ◽  
Control Flight ◽  
Female Specific

AbstractAedes aegypti is a vector of dengue, chikungunya, and Zika viruses. Current vector control strategies such as community engagement, source reduction, and insecticides have not been sufficient to prevent viral outbreaks. Thus, interest in novel strategies involving genetic engineering is growing. Female mosquitoes rely on flight to mate with males and obtain a bloodmeal from a host. We hypothesized that knockout of genes specifically expressed in female mosquitoes associated with the indirect flight muscles would result in a flightless female mosquito. With the CRISPR-Cas9 system, we performed embryonic microinjections of Cas9 protein and guide RNAs specific to genes hypothesized to control flight in mosquitoes, and have obtained genetic knockouts in several genes specifically expressed in the flight-muscle, including those specific to female flight muscle. Analysis of the phenotype of these female-specific gene knockout mutants resulted in flightless females and flying males. While further assessment is required, this work lays the groundwork for a mechanism of population control that is female-specific for the Ae. aegypti vector.

scholarly journals The Developmental Transcriptome of Aedes albopictus, a Major Worldwide Human Disease Vector

2020 ◽  
Vol 10 (3) ◽  
pp. 1051-1062 ◽  
Author(s):  
Stephanie Gamez ◽  
Igor Antoshechkin ◽  
Stelia C. Mendez-Sanchez ◽  
Omar S. Akbari
Keyword(s):  
Gene Expression ◽  
Aedes Aegypti ◽  
Aedes Albopictus ◽  
Human Disease ◽  
Control Strategies ◽  
Expression Profiles ◽  
Expression Patterns ◽  
Disease Vector ◽  
Biological Studies ◽  
Similarities And Differences

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.

scholarly journals A functional requirement for sex-determination M/m locus region lncRNA genes in Aedes aegypti female larvae

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Keshava Mysore ◽  
Limb K. Hapairai ◽  
Ping Li ◽  
Joseph B. Roethele ◽  
Longhua Sun ◽  
...  
Keyword(s):  
Aedes Aegypti ◽  
Sex Determination ◽  
Population Control ◽  
Control Strategies ◽  
Adult Males ◽  
Non Coding Rna ◽  
Female Specific ◽  
Interfering Rna ◽  
Long Non Coding Rna ◽  
Male Mosquitoes

AbstractAlthough many putative long non-coding RNA (lncRNA) genes have been identified in insect genomes, few of these genes have been functionally validated. A screen for female-specific larvicides that facilitate Aedes aegypti male sex separation uncovered multiple interfering RNAs with target sites in lncRNA genes located in the M/m locus region, including loci within or tightly linked to the sex determination locus. Larval consumption of a Saccharomyces cerevisiae (yeast) strain engineered to express interfering RNA corresponding to lncRNA transcripts resulted in significant female death, yet had no impact on male survival or fitness. Incorporation of the yeast larvicides into mass culturing protocols facilitated scaled production and separation of fit adult males, indicating that yeast larvicides could benefit mosquito population control strategies that rely on mass releases of male mosquitoes. These studies functionally verified a female-specific developmental requirement for M/m locus region lncRNA genes, suggesting that sexually antagonistic lncRNA genes found within this highly repetitive pericentromeric DNA sequence may be contributing to the evolution of A. aegypti sex chromosomes.

scholarly journals Antiviral Effectors and Gene Drive Strategies for Mosquito Population Suppression or Replacement to Mitigate Arbovirus Transmission by Aedes aegypti

Insects ◽  
2020 ◽  
Vol 11 (1) ◽  
pp. 52 ◽  
Author(s):  
Adeline Williams ◽  
Alexander Franz ◽  
William Reid ◽  
Ken Olson
Keyword(s):  
Aedes Aegypti ◽  
Disease Transmission ◽  
Mosquito Control ◽  
Control Strategies ◽  
Mosquito Vector ◽  
Close Monitoring ◽  
Gene Drive ◽  
Population Replacement ◽  
Effector Genes ◽  
Population Suppression

The mosquito vector Aedes aegypti transmits arthropod-borne viruses (arboviruses) of medical importance, including Zika, dengue, and yellow fever viruses. Controlling mosquito populations remains the method of choice to prevent disease transmission. Novel mosquito control strategies based on genetically manipulating mosquitoes are being developed as additional tools to combat arbovirus transmission. Genetic control of mosquitoes includes two basic strategies: population suppression and population replacement. The former aims to eliminate mosquito populations while the latter aims to replace wild populations with engineered, pathogen-resistant mosquitoes. In this review, we outline suppression strategies being applied in the field, as well as current antiviral effector genes that have been characterized and expressed in transgenic Ae. aegypti for population replacement. We discuss cutting-edge gene drive technologies that can be used to enhance the inheritance of effector genes, while highlighting the challenges and opportunities associated with gene drives. Finally, we present currently available models that can estimate mosquito release numbers and time to transgene fixation for several gene drive systems. Based on the recent advances in genetic engineering, we anticipate that antiviral transgenic Ae. aegypti exhibiting gene drive will soon emerge; however, close monitoring in simulated field conditions will be required to demonstrate the efficacy and utility of such transgenic mosquitoes.

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