2025
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Tepper, K., King, J., Cholan, P.M., Pfitzner, C., Morsch, M., Apte, S.C., Maselko, M. Methylmercury demethylation and volatilization by animals expressing microbial enzymes. Nat Commun 16, 1117 (2025).
Mercury is a highly toxic trace metal that readily biomagnifies in food webs where it is inaccessible to current bioremediation methods. Animals could potentially be engineered to detoxify mercury within their food webs to clean up impacted ecosystems. We demonstrate that invertebrate (Drosophila melanogaster) and vertebrate (Danio rerio) animal models can express organomercurial lyase (MerB) and mercuric reductase (MerA) from Escherichia coli to demethylate methylmercury and remove it from their biomass as volatile elemental mercury. The engineered animals accumulated less than half as much mercury relative to their wild-type counterparts, and a higher proportion of mercury in their tissue was in the form of less bioavailable inorganic mercury. Furthermore, the engineered animals could tolerate higher exposures to methylmercury compared to controls. These findings demonstrate the potential of using engineered animals for bioremediation and may be applied to reduce the burden of methylmercury in impacted ecosystems by disrupting its biomagnification or to treat contaminated organic waste streams. |
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Beach, SJ., Maselko, M. Recombinant Venom Proteins in Insect Seminal Fluid Reduce Female Lifespan. Nature Communications 16, no. 1 (7 January 2025): 219.
The emergence of insecticide resistance has increased the need for alternative pest management tools. Numerous genetic biocontrol approaches, which involve the release of genetically modified organisms to control pest populations, are in various stages of development to provide highly targeted pest control. However, all current mating-based genetic biocontrol technologies function by releasing engineered males which skew sex-ratios or reduce offspring viability in subsequent generations which leaves mated females to continue to cause harm (e.g. transmit disease). Here, we demonstrate intragenerational genetic biocontrol, wherein mating with engineered males reduces female lifespan. The toxic male technique (TMT) involves the heterologous expression of insecticidal proteins within the male reproductive tract that are transferred to females via mating. In this study, we demonstrate TMT in Drosophila melanogaster males, which reduce the median lifespan of mated females by 37 − 64% compared to controls mated to wild type males. Agent-based models of Aedes aegypti predict that TMT could reduce rates of blood feeding by a further 40 – 60% during release periods compared to leading biocontrol technologies like fsRIDL. TMT is a promising approach for combatting outbreaks of disease vectors and agricultural pests. |
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2024
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Retief C, Kumar S, Tepper K, Maselko M. Biomanufacturing of a functional microbial phytase in an insect host. Journal of Insects as Food and Feed. 2024 Dec 13;1(aop):1-0.
Insects, such as Black Soldier Flies (Hermetia illucens), are increasingly used as sustainable animal feed ingredients that can be reared on plentiful organic substrates such as agricultural residues and pre-consumer food waste. Genetically engineering insects to heterologously express feed additive enzymes has the potential to generate more value from organic waste, while improving livestock health and productivity. Phytases are widely used feed additive enzymes that hydrolyse the phosphate groups from the myo-inositol backbone of phytic acid, a phosphate rich antinutrient compound that monogastric animals cannot efficiently digest. Dietary phytase supplementation improves absorption of phosphorous, proteins, and cationic nutrients, while mitigating the negative environmental effects of phytic acid rich excreta. We evaluated the potential of using insects to biomanufacture microbial feed additive enzymes by engineering the model insect, Drosophila melanogaster, to express phytases. One histidine acid phytase, three beta propellor phytases, three purple acid phosphatases, and one PTP-like phytase were selected for screening in D. melanogaster. Transgenic flies expressing the AppA histidine acid phytase from E. coli had 27.82 FTU/g of phytase activity, which exceeds the 0.5-1.0 FTU/g required in animal feed. Maximum activity from AppA phytase expressed by D. melanogaster was observed at pH 5 and 55 °C, however, more than 50% of phytase activity was present at 25 °C and pH 2. Here we demonstrate that insects may be suitable hosts for the heterologous expression of a microbial phytase enzyme with applications for improving animal feed nutrition and organic waste valorisation. |
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Clark M, Nguyen C, Nguyen H, Tay A, Beach SJ, Maselko M, López Del Amo V. Expanding the CRISPR base editing toolbox in Drosophila melanogaster. Communications Biology. 2024 Sep 12;7(1):1126.
CRISPR base editors can introduce point mutations into DNA precisely, and cytosine base editors (CBEs) catalyze C to T transitions. While CBEs have been thoroughly explored in cell culture and organisms such as mice, little is known about DNA base editing in insects. In this study, we evaluated germline editing rates of three different CBEs expressed under actin (ubiquitous) or nanos (germline) promoters utilizing Drosophila melanogaster. The original Rattus norvegicus-derived cytosine deaminase APOBEC1 (rAPO-1) displayed high base editing rates (~99%) with undetectable indel formation. Additionally, we show that base editors can be used for generating male sterility and female lethality. Overall, this study highlights the importance of promoter choice and sex-specific transmission for efficient base editing in flies while providing new insights for future genetic biocontrol designs in insects. |
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Nguyen H, Nguyen B, Mainali B, Maselko M. Optimization of rearing Transeius montdorensis under laboratory conditions. bioRxiv. 2024:2024-09.
The global application of Transeius montdorensis (Acari: Phytoseiidae) as a biological control agent across various protected crops has proven effective against a range of insect pests like thrips and whiteflies, as well as pest mites like broad mites and russet mites. Optimization of rearing T. montdorensis under laboratory conditions is crucial for further studies of this species to improve their application in Integrated Pest Management (IPM) programs. Here, we evaluated the development and reproduction of T. montdorensis when fed on four different diets, including cattail pollen (Typha latifolia), living dried fruit mites (Carpoglyphus lactis), frozen C. lactis eggs, and a mixed diet of frozen C. lactis eggs and T. latifolia pollen. Females consuming the mixed diet exhibited superior total fecundity and daily oviposition rate, along with the highest intrinsic rate of increase (rm) and net productive rate (R0) among all diets tested. The immature period was significantly longer for mites on a diet of living C. lactis compared to those on other diets. Importantly, utilizing frozen C. lactis eggs and T. latifolia pollen mitigates the risk of infestation and contamination from the living dried fruit mites, which is important for laboratory and field settings when releasing the predator colonies. Our findings not only present an optimized rearing method for predatory mites under laboratory conditions but also suggest potential broader applications for enhancing the effectiveness and sustainability of biological control strategies across various agroecosystems and reducing dependency on chemical pesticides. |
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Tay AP, Didi K, Wickramarachchi A, Bauer DC, Wilson LO, Maselko M. Synsor: a tool for alignment-free detection of engineered DNA sequences. Frontiers in Bioengineering and Biotechnology. 2024 Jul 12;12:1375626.
DNA sequences of nearly any desired composition, length, and function can be synthesized to alter the biology of an organism for purposes ranging from the bioproduction of therapeutic compounds to invasive pest control. Yet despite offering many great benefits, engineered DNA poses a risk due to their possible misuse or abuse by malicious actors, or their unintentional introduction into the environment. Monitoring the presence of engineered DNA in biological or environmental systems is therefore crucial for routine and timely detection of emerging biological threats, and for improving public acceptance of genetic technologies. To address this, we developed Synsor, a tool for identifying engineered DNA sequences in high-throughput sequencing data. Synsor leverages the k-mer signature differences between naturally occurring and engineered DNA sequences and uses an artificial neural network to classify whether a DNA sequence is natural or engineered. By querying suspected sequences against the model, Synsor can identify sequences that are likely to have been engineered. Using natural plasmid and engineered vector sequences, we showed that Synsor identifies engineered DNA with >99% accuracy. We demonstrate how Synsor can be used to detect potential genetically engineered organisms and locate where engineered DNA is being introduced into the environment by analysing genomic and metagenomic data from yeast and wastewater samples, respectively. Synsor is therefore a powerful tool that will streamline the process of identifying engineered DNA in poorly characterized biological or environmental systems, thereby allowing for enhanced monitoring of emerging biological threats. |
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Tepper K, Edwards O, Sunna A, Paulsen IT, Maselko M. Diverting organic waste from landfills via insect biomanufacturing using engineered black soldier flies (Hermetia illucens). Communications Biology. 2024 Jul 24;7(1):862.
A major roadblock towards the realisation of a circular economy are the lack of high-value products that can be generated from waste. Black soldier flies (BSF; Hermetia illucens) are gaining traction for their ability to rapidly consume large quantities of organic wastes. However, these are primarily used to produce a small variety of products, such as animal feed ingredients and fertiliser. Using synthetic biology, BSF could be developed into a novel sustainable biomanufacturing platform to valorise a broader variety of organic waste feedstocks into enhanced animal feeds, a large variety of high-value biomolecules including industrial enzymes and lipids, and improved fertiliser. |
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Johnson ML, Hay B, Maselko M. Allele Sails: launching traits and fates into wild populations with DNA sequence modifiers. Nature Communications 15, 6665 (2024).
Population-scale genome editing can be used to alter the composition or fate of wild populations. One approach to achieving these aims utilizes a synthetic gene drive element—a multi-gene cassette—to bring about an increase in the frequency of an existing allele. However, the use of gene drives is complicated by the multiple scientific, regulatory, and social issues associated with transgene persistence and gene flow. Alternatives in which transgenes are not driven could potentially avoid some of these issues. Here we propose an approach to population scale gene editing using a system we refer to as an Allele Sail. An Allele Sail consists of a genome editor (the Wind) that introduces DNA sequence edits (the Sail) at one or more sites, resulting in progeny that are viable and fertile. The editor, such as a sequence-specific nuclease, or a prime- or base-editor, is inherited in a Mendelian fashion. Meanwhile, the edits it creates experience a Super-Mendelian increase in frequency. We explore this system using agent-based modeling, and identify contexts in which a single, low frequency release of an editor brings edits to a very high frequency. We also identify conditions in which manipulation of sex determination can be used to bring about population suppression. Current regulatory frameworks often distinguish between transgenics as genetically modified organisms (GMOs), and their edited non-transgenic progeny as non-GMO. In this context an Allele Sail provides a path to alter traits and fates of wild populations in ways that may be considered more acceptable. |
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Pfitzner C, Tepper K, Kumar S, Retief C, McNab J, Harrell RA, Maselko M. Rapid generation and screening of transgenic black soldier fly (Hermetia illucens). bioRxiv. 2024:2024-02.
Background The black soldier fly (BSF), Hermetia illucens is a widely used, and mass-produced insect that fulfils an important role in both the management of organic waste and as a component of animal feed formulations. They also have significant potential as a platform for converting organic waste into high-value proteins, and lipids for the production of biofuels. Applying synthetic biology to BSF provides even more potential for improvement through the generation of transgenic BSF to enhance animal feed, produce and fine tune high-value industrial biomolecules, and to expand their waste conversion capabilities. Results To enable the rapid generation and screening of transgenic BSF, we utilised microinjections of piggyBac mRNA with donor plasmids. We have found preliminary screening of G0 BSF to identify mosaics for outcrossing can be completed less than 2 weeks after microinjection. Stable transgenic lines were reliably generated with effective transformation rates of 30-33%, and transmission of the transgene could be confirmed 3 days after outcrossing the G0 adults. We also present a protocol for identifying the location of integrated transgenes. Conclusions The methods presented here expedite the screening process for BSF transgenesis and further expand the toolkit for BSF synthetic biology. |
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2022
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Upadhyay A, Feltman NR, Sychla A, Janzen A, Das SR, Maselko M, Smanski M. Genetically engineered insects with sex-selection and genetic incompatibility enable population suppression. Elife. 2022 Feb 2;11:e71230.
Engineered Genetic Incompatibility (EGI) is a method to create species-like barriers to sexual reproduction. It has applications in pest control that mimic Sterile Insect Technique when only EGI males are released. This can be facilitated by introducing conditional female-lethality to EGI strains to generate a sex-sorting incompatible male system (SSIMS). Here, we demonstrate a proof of concept by combining tetracycline-controlled female lethality constructs with a pyramus-targeting EGI line in the model insect Drosophila melanogaster. We show that both functions (incompatibility and sex-sorting) are robustly maintained in the SSIMS line and that this approach is effective for population suppression in cage experiments. Further we show that SSIMS males remain competitive with wild-type males for reproduction with wild-type females, including at the level of sperm competition. |
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2021
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Clark, M., Tepper, K., Petroll, K., Kumar, S., Sunna, A. and Maselko, M. Bioremediation of industrial pollutants by insects expressing a fungal laccase. ACS Synthetic Biology. 2021 Dec 9;11(1):308-16.
Inadequate management of household and industrial wastes poses major challenges to human and environmental health. Advances in synthetic biology may help address these challenges by engineering biological systems to perform new functions such as biomanufacturing of high-value compounds from low-value waste streams and bioremediation of industrial pollutants. The current emphasis on microbial systems for biomanufacturing, which often requires highly preprocessed inputs and sophisticated infrastructure, is not feasible for many waste streams. Furthermore, concerns about transgene biocontainment have limited the release of engineered microbes or plants for bioremediation. Engineering of animals may provide opportunities for utilizing various waste streams that are not suitable for microbial biomanufacturing while effective transgene biocontainment options should enable in situ bioremediation. Here, we engineer the model insect Drosophila melanogaster to express a functional laccase from the fungus Trametes trogii. Laccase-expressing flies reduced concentrations of the endocrine disruptor bisphenol A by more than 50% when present in their growth media. A lyophilized powder prepared from engineered adult flies retained substantial enzymatic activity, degrading more than 90% of bisphenol A and the textile dye indigo carmine in aqueous solutions. Our results demonstrate that transgenic animals may be used to bioremediate environmental contaminants in vivo and serve as novel production platforms for industrial enzymes. These results support further development of insects, and possibly other animals, as bioproduction platforms and their potential use in bioremediation. |
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2020
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Das SR, Maselko M, Upadhyay A, Smanski MJ. Genetic engineering of sex chromosomes for batch cultivation of non-transgenic, sex-sorted males. PLoS genetics. 2020 Nov 2;16(11):e1009180.
The field performance of Sterile Insect Technique (SIT) is improved by sex-sorting and releasing only sterile males. This can be accomplished by resource-intensive separation of males from females by morphology. Alternatively, sex-ratio biasing genetic constructs can be used to selectively remove one sex without the need for manual or automated sorting, but the resulting genetically engineered (GE) control agents would be subject to additional governmental regulation. Here we describe and demonstrate a genetic method for the batch production of non-GE males. This method could be applied to generate the heterogametic sex (XY, or WZ) in any organism with chromosomal sex determination. We observed up to 100% sex-selection with batch cultures of more than 103 individuals. Using a stringent transgene detection assay, we demonstrate the potential of mass production of transgene free males. |
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Maselko M, Feltman N, Upadhyay A, Hayward A, Das S, Myslicki N, Peterson AJ, O’Connor MB, Smanski MJ. Engineering multiple species-like genetic incompatibilities in insects. Nature communications. 2020 Sep 8;11(1):4468.
Speciation constrains the flow of genetic information between populations of sexually reproducing organisms. Gaining control over mechanisms of speciation would enable new strategies to manage wild populations of disease vectors, agricultural pests, and invasive species. Additionally, such control would provide safe biocontainment of transgenes and gene drives. Here, we demonstrate a general approach to create engineered genetic incompatibilities (EGIs) in the model insect Drosophila melanogaster. EGI couples a dominant lethal transgene with a recessive resistance allele. Strains homozygous for both elements are fertile and fecund when they mate with similarly engineered strains, but incompatible with wild-type strains that lack resistant alleles. EGI genotypes can also be tuned to cause hybrid lethality at different developmental life-stages. Further, we demonstrate that multiple orthogonal EGI strains of D. melanogaster can be engineered to be mutually incompatible with wild-type and with each other. EGI is a simple and robust approach in multiple sexually reproducing organisms. |
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Clark M, Maselko M. Transgene biocontainment strategies for molecular farming. Frontiers in Plant Science. 2020 Mar 3;11:210.
Advances in plant synthetic biology promise to introduce novel agricultural products in the near future. ‘Molecular farms’ will include crops engineered to produce medications, vaccines, biofuels, industrial enzymes, and other high value compounds. These crops have the potential to reduce costs while dramatically increasing scales of synthesis and provide new economic opportunities to farmers. Current transgenic crops may be considered safe given their long-standing use, however, some applications of molecular farming may pose risks to human health and the environment. Unwanted gene flow from engineered crops could potentially contaminate the food supply, and affect wildlife. There is also potential for unwanted gene flow into engineered crops which may alter their ability to produce compounds of interest. Here, we briefly discuss the applications of molecular farming and explore the various genetic and physical methods that can be used for transgene biocontainment. As yet, no technology can be applied to all crop species, such that a combination of approaches may be necessary. Effective biocontainment is needed to enable large scale molecular farming. |
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2019
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Bajer PG, Ghosal R, Maselko M, Smanski MJ, Lechelt JD, Hansen G, Kornis MS. Biological control of invasive fish and aquatic invertebrates: a brief review with case studies. Management of Biological Invasions. 2019 Jun 1;10(2):227.
We review various applications of biocontrol for invasive fish and aquatic invertebrates. We adopt a broader definition of biocontrol that includes traditional methods like predation and physical removal (biocontrol by humans), and modern approaches like genetic engineering and use of microbes (including pathogens). While physical removal and predation (by native predators) are used relatively commonly, use of genetic technologies and microbes is in developmental stages. The two latter strategies are most advanced in case of the common carp (Cyprinus carpio), one of the world’s most invasive fish; virus release to control carp might soon occur in Australia. Drawing from empirical examples in North America, we emphasize that biocontrol strategies are most likely to be successful if they include multiple approaches that target specific behaviors or weaknesses in pests’ life histories. This is illustrated by reviewing case studies on the common carp and rusty crayfish (Orconectes rusticus) in Midwestern North America. In case of the common carp, basic research on movement patterns and recruitment bottlenecks identified a strategy where winter aggregations of adults were targeted for removal with nets, while native predators of carp eggs and larvae were instrumental in controlling carp’s reproductive success. In the case of the rusty crayfish, basic research on interactions between crayfish, habitat, and native predators identified a successful strategy of stocking selected native predators to control juvenile crayfish in conjunction with physical removal of adult crayfish using traps. We are also reviewing the case of the round goby (Neogobius melanostomus) in the Great Lakes. In this example, multiple pieces of evidence (diet, bioenergetics) illustrate how initially abundant pest was brought under control (in some areas) by several species of native predators in a large, natural ecosystem. Overall, examples of successful biocontrol of aquatic pests have been rare and have relied on physical removal and predation. We expect that new technologies (e.g. genetic technologies) will occur in the next decade but will have to clear regulatory and ethical concerns before they are applied. While developing more sophisticated control techniques, we advocate for more basic research on the life history of the pests to identify behavioral or developmental weaknesses that could be targeted with specific tools to increase chances of success while minimizing impacts on native ecosystems. |
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2017
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Maselko M, Heinsch SC, Chacón JM, Harcombe WR, Smanski MJ. Engineering species-like barriers to sexual reproduction. Nature communications. 2017 Oct 12;8(1):883.
Controlling the exchange of genetic information between sexually reproducing populations has applications in agriculture, eradication of disease vectors, control of invasive species, and the safe study of emerging biotechnology applications. Here we introduce an approach to engineer a genetic barrier to sexual reproduction between otherwise compatible populations. Programmable transcription factors drive lethal gene expression in hybrid offspring following undesired mating events. As a proof of concept, we target the ACT1 promoter of the model organism Saccharomyces cerevisiae using a dCas9-based transcriptional activator. Lethal overexpression of actin results from mating this engineered strain with a strain containing the wild-type ACT1 promoter. |
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