Biology subjects

Smanski, M.

Publications and source records attributed to Smanski, M..

10 recordsLinked to original sources

bioRxiv2025

Improving Perennial Ryegrass Transformation Protocols with Developmental Regulators

The development of genetically modified (GM) or gene edited (GE) turfgrass requires transformation systems that are both efficient and broadly applicable across genotypes. However, traditional Agrobacterium-mediated callus culture methods remain limited by low transformation efficiency, extended culture durations, and strong genotype dependence. In this study, we compare a modified classical callus culture protocol with an approach that incorporates the developmental regulator genes WUSCHEL2 (wus2) and BABY BOOM (bbm), along with an inducible Cre recombinase and a dual luciferase assay to test variable promoter strengths in perennial ryegrass (Lolium perenne L.). We show that traditional protocols failed to regenerate plants, despite successful callus formation and transgene expression. In contrast, the developmental regulator system enabled efficient callus induction and plant regeneration independent of genotype. This optimized protocol significantly reduces the time and genotype constraints of perennial ryegrass transformation, offering a practical platform for advanced genetic engineering applications of an important turf and forage grass.

plant biology↗

bioRxiv2025

Heterologous expression of microbial nitroreductases for TNT degradation in transgenic animals

TNT (2,4,6-trinitrotoluene) from unexploded ordinances is a common environmental pollutant near munitions factories, military training sites, and areas of armed conflict. As physical and chemical approaches for TNT remediation are costly and difficult to scale, in situ bioremediation is an attractive alternative. TNT is a phytoaccumulative pollutant. Herbivores engineered to detoxify TNT are a potentially cost-effective platform to bioremediate large areas of contaminated sites while grazing or browsing. As a first step towards engineering herbivores with metabolic capabilities for TNT degradation, we engineered the animal genetic model, Drosophila melanogaster, to screen a library of microbial nitroreductases that can catalyse the initial degradation steps required for TNT degradation. We find strong, cofactor-dependent activity in fly lysates engineered to express NsfA from Escherichia coli, and NsfI from Enterobacter cloacae.

synthetic biology↗

bioRxiv2024

The strength of selection for costly toxin production increases with growth rate.

Microbes adopt a diversity of strategies to successfully compete with coexisting strains for space and resources. One common strategy is the production of toxic compounds to inhibit competitors, but the strength and direction of selection for this strategy varies depending on the environment. In particular, existing theoretical and experimental evidence suggests growth in spatially-structured environments makes toxin production more beneficial because competitive interactions are localized. Because higher growth rates increase the localization of resource competition in a structured environment, theory predicts that toxin production should be especially beneficial under these conditions. We tested this hypothesis by developing a genome-scale metabolic modeling approach and complementing it with comparative genomics to investigate the impact of growth rate on selection for costly toxin production. Our modeling approach expands the current abilities of the dynamic flux balance analysis platform COMETS to incorporate signaling and toxin production. Using this capability, we find that our modeling framework predicts that the strength of selection for toxin production increases as growth rate increases. This finding is supported by comparative genomics analyses that include diverse microbial species. Our work emphasizes that toxin production is more likely to be maintained in rapidly-growing, spatially-structured communities, thus improving our ability to manage critical microbial communities and informing natural product discovery.

evolutionary biology↗

bioRxiv2024

High-throughput investigation of genetic design constraints in domesticated Influenza A Virus for transient gene delivery

Replication-incompetent single cycle infectious Influenza A Virus (sciIAV) has demonstrated utility as a research and vaccination platform. Protein-based therapeutics are increasingly attractive due to their high selectivity and potent efficacy but still suffer from low bioavailability and high manufacturing cost. Transient RNA-mediated delivery is a safe alternative that allows for expression of protein-based therapeutics within the target cells or tissues but is limited by delivery efficiency. Here, we develop recombinant sciIAV as a platform for transient gene delivery in vivo and in vitro for therapeutic, research, and manufacturing applications (in vivo antimicrobial production, cell culture contamination clearance, and production of antiviral proteins in vitro). While adapting the system to deliver new protein cargo we discovered expression differences presumably resulting from genetic context effects. We applied a high-throughput screen to map these within the 3'-untranslated and coding regions of the hemagglutinin-encoding segment 4. This screen revealed permissible mutations in the 3'-UTR and depletion of RNA level motifs in the N-terminal coding region.

bioengineering↗

bioRxiv2023

Perceptions on the genetic biocontrol of invasive carp

Common carp (Cyprinus carpio) is an invasive species that damages native aquatic ecosystems. Advances in genetic engineering have led to novel genetic biocontrol methods that have the potential to aid in the control of carp and other invasive species. Research and development of these technologies is proceeding in parallel with outreach activities which aim to inform and engage the public. Public perceptions on the ethics, economics, and policy decisions surrounding the possible application of genetic control technologies are important in decision-making processes. Here we report results from a survey of more than 1,300 adults that measures the perceptions of risks, benefits, and overall comfort with a panel of existing and proposed carp control methods. Our population sample largely agrees that carp control is an important issue that demands action. We observe trends suggesting that comfort levels with the possible future implementation of genetic biocontrol for common carp are strongly influenced by the perception of benefits compared to other methods, in particular effectiveness and environmental safety. No statistically significant correlations between comfort levels and river/lake use habits nor demographics are observed. There is a need for further research to identify how respondents from similar backgrounds come to different knowledge and attitudes on the matter.

scientific communication and education↗

bioRxiv2023

Efficient gene activation in plants by the MoonTag programmable transcriptional activator

CRISPR/Cas-based transcriptional activators have been developed to induce gene expression in eukaryotic and prokaryotic organisms. The main advantages of CRISPR-Cas based systems is that they can achieve high levels of transcriptional activation and are very easy to program via pairing between the guide RNA and the DNA target strand. SunTag is a second-generation system that activates transcription by recruiting multiple copies of an activation domain (AD) to its target promoters. SunTag is a strong activator; however, in some species it is difficult to stably express. To overcome this problem, we designed MoonTag, a new activator that worked on the same basic principle as SunTag, but whose components are better tolerated when stably expressed in transgenic plants. We demonstrate that MoonTag is capable of inducing high levels of transcription in all plants tested. In Setaria, MoonTag is capable of inducing high levels of transcription of reporter genes as well as of endogenous genes. More important, MoonTag components are expressed in transgenic plants to high levels without any deleterious effects. MoonTag is also able to efficiently activate genes in eudicotyledonous species such as Arabidopsis and tomato. Finally, we show that MoonTag activation is functional across a range of temperatures, which is promising for potential field applications.

bioengineering↗

bioRxiv2022

Optimized dCas9 Transcription Activators for Plants

Understanding how gene expression impacts plant development and physiology is important for rationally engineering improved crops. Programmable Transcriptional Activators (PTAs), including CRISPR-Cas activators, have traditionally relied on a limited number of transcriptional activation domains. Usually the VP64 domain, derived from human herpes simplex virus, is fused to a DNA-binding domain to activate target gene expression. We reasoned there was considerable space for PTA improvement by replacing VP64 with a plant-derived activation domain. To address this, we designed, built, and tested a PTA library of 38 putative plant transcriptional activation domains. Domains from HSFA6b, AvrXa10, DOF1, DREB1, and DREB2 genes function as strong activators in Setaria viridis and Arabidopsis thaliana in protoplast assays and in transgenic plants. Overexpression of multiple endogenous genes (FT, PAP1, WUS) reached levels similar to the highly expressed housekeeping gene, PP2A, regardless of basal expression level. Further, these domains were effective in different PTA architectures, including the dCas9-SunTag, dCas9-Moontag, and TALE-SunTag systems. Lastly, we demonstrate the ability of these improved PTAs to map enhancer regions that promote gene expression in plants. This work demonstrates the effective and flexible nature of PTAs to activate target genes in plants, providing tools that can be used to improve agronomically relevant traits of interest.

plant biology↗

bioRxiv2021

Genetically engineered insects with sex-selection and genetic incompatibility enable population suppression

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.

bioengineering↗

bioRxiv2020

Engineering multiple species-like genetic incompatibilities in insects

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. Natural speciation can be driven by pre-zygotic barriers that prevent fertilization or by post-zygotic genetic incompatibilities that render the hybrid progeny inviable or sterile. Here we demonstrate a general approach to create engineered genetic incompatibilities (EGIs) in the model insect Drosophila melanogaster. Our system couples a dominant lethal transgene with a recessive resistance allele. EGI strains that are 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. We show that EGI genotypes can 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. Our approach to create EGI organisms is simple, robust, and functional in multiple sexually reproducing organisms.

synthetic biology↗

bioRxiv2019

Batch cultivation of non-transgenic males suitable for SIT programs

The field performance of Sterile Insect Technique (SIT) is improved by sex-sorting and releasing only the 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 kill 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 method for the batch production of non-GE males that is applicable for sex-selective production of males suitable for genetic biocontrol programs. 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 rearing of transgene free males.

synthetic biology↗