Biology subjects

Galizi, R.

Publications and source records attributed to Galizi, R..

11 recordsLinked to original sources

bioRxiv2026

Ontology-driven software engineering using LLMs for knowledge graphs in engineering biology

Large language models have transformed software engineering practices. However, generated artefacts are not always developer-friendly and may partially meet complex requirements. As the need to standardise, integrate, and develop tools in engineering biology increases, novel approaches are needed to create and maintain intuitive software sustainably. Here, we present an ontology-driven approach using large language models to create user-facing software libraries for knowledge graphs. We introduce an ontology-to-language framework to systematically map domain terms and graph structures. We then demonstrate this approach by creating an ontology for the latest Synthetic Biology Open Language standard and generating the sbol-script software library, which can be used within browsers or to develop applications with native web support. This ontology-driven software engineering approach and these resources are essential for the community and to facilitate the development of sustainable software projects. The SBOL3 Ontology and the sbol-script library are available from https://github.com/SynBioDex/sbol-owl3 and https://github.com/SynBioDex/sbol-script.

synthetic biology↗

bioRxiv2026

The Y chromosome gene draupnir reveals constraints on engineering Y-linked sex-ratio distorters in malaria mosquitoes

Y chromosome sex distorters offer a powerful strategy for mosquito population suppression, but their implementation is constrained by the transcriptional silencing of Y-linked transgenes during spermatogenesis. To investigate how endogenous Y genes evade this repression, we characterized draupnir (formerly YG5), a multicopy Y-linked gene of Anopheles gambiae encoding a Zip3-like meiotic protein. Comparative genomic and phylogenetic analyses revealed that draupnir originated through duplication of the autosomal paralog skirnir, itself derived from the ancestral recombination factor vilya, followed by amplification into a tandem array on the Y chromosome. We show that draupnir is actively transcribed during meiosis, making it the only known Y-linked gene expressed during mosquito spermatogenesis. To test whether its regulatory region is sufficient to confer sperm-specific expression from the Y chromosome, we cloned the draupnir promoter to drive expression of an X-chromosome shredder. When inserted on an autosome, the construct drove meiotic expression and strong sex-ratio distortion in the progeny of transgenic males. In contrast, the same construct inserted on the Y chromosome was transcriptionally silent and produced balanced sex ratios. These results demonstrate that draupnir expression depends on its native genomic context within a multicopy Y-linked array and that its promoter alone cannot overcome transcription silencing. Our findings reveal a fundamental constraint on Y-chromosome-based genetic control strategies and point to future approaches for enabling transcription from otherwise repressed sex chromosomes.

genetics↗

bioRxiv2026

A genetically encoded local learning rule enables physical learning in engineered bacteria

Training physical neural networks directly in matter remains difficult because most platforms do not store and update weights within the same physical substrate. Here we show that engineered Escherichia coli can implement a genetically encoded local learning rule acting on persistent biological memory. We introduce memregulons: bacterial memory elements in which coupled plasmids store an analogue weight as the relative abundance of P1 within the P1+P2 plasmid pool. Environmental inputs activate local promoters, and a shared sublethal kanamycin signal converts promoter activity into differential growth that lowers the stored P1 fraction. In single strains, flow-cytometry trajectories across distinct promoters support the predicted dependence of weight change on learning-channel activity and standing population variance. At the single-cell level, repeated negative learning reshapes the stored weight distribution as it approaches the lower boundary. In mixed populations and co-cultures, one shared negative learning signal selectively rewrites active memregulons, enabling externally routed supervised tic-tac-toe lessons. We then generalise the architecture across orthogonal chemical inputs and combinatorial promoters, and use experimentally measured updates in hybrid analyses of winner-take-all and nonlinear-classifier tasks. These results establish physical learning in living matter through local negative updates of genetically encoded analogue weights.

synthetic biology↗

bioRxiv2025

Microsatellite Analysis of Genetic Differentiation Among Populations of the Malaria Vector, Anopheles funestus, across Mainland Tanzania Reveals Contrasted Patterns of Geographic Isolation and Gene Flow

BackgroundDespite Anopheles funestus s.s. being a highly competent and widespread malaria vector in Africa, its population structure remains largely understudied in many countries, including Tanzania. Herein, we examine the genetic diversity, geographic isolation, and gene flow of An. funestus populations across 10 administrative regions in mainland Tanzania. MethodsWe employed 12 previously used microsatellite DNA markers to describe genetic diversity, isolation by distance, and gene flow patterns among 10 An. funestus s.s. populations (n = 654) and one An. parensis population (n = 28), used as an outgroup, sampled across 10 regions in mainland Tanzania. ResultsOverall, allelic richness (Na) and genetic diversity (HS) did not differ significantly among populations. Although some loci and populations showed significant departures from Hardy-Weinberg equilibrium, the patterns were not indicative of sub-structuring within locations. Pairwise genetic divergence (FST) values indicated clear separation between An. parensis and An. funestus s.s., with values exceeding 0.2, consistent with species-level differentiation. Among An. funestus s.s. populations, the highest divergence was observed between southeastern coastal populations (Mtwara, Ruvuma, Lindi) and inland populations, with FST values up to 0.288. There was no evidence of isolation by distance. Instead, patterns of genetic divergence suggested connectivity across the Rift Valley and heterogeneity among southeastern populations. Neighbour-joining analysis and Bayesian genotype clustering identified three distinct population groups: (i) An. parensis (Dodoma), (ii) a genetically distinct An. funestus s.s. population from Mtwara, and (iii) a more homogeneous cluster comprising the remaining An. funestus s.s. populations. Notably, the Mtwara population appeared highly differentiated, with divergence approaching that between An. Funestus s.s. and An. parensis, supporting its distinctiveness but not undermining the role of An. parensis as an outgroup. ConclusionExcept for the Mtwara population, whose status will need to be clarified through whole genome sequencing, moderate genetic divergence was found among An. funestus s.s. populations across Tanzania, despite geographical separation and the Rift Valley. The observed genetic structure suggests that anthropogenic gene flow may play a key role in shaping population divergence. Future studies should aim to delineate the effects of local adaptation from recent gene flow to further explore these dynamics.

genetics↗

bioRxiv2022

Quantification of genetic variants in bacterial cultures by Sanger sequencing

Genetic variations such as mutations and recombinations arise spontaneously in all cultured organisms. Although it is possible to identify non-neutral mutations by selection or counter- selection, neutral mutations usually require DNA sequencing to be identified in a population, which are normally expensive and time-consuming. Neutral mutations could even become dominant under changing environmental conditions enforcing transitory selection or counter- selection. We propose a novel methodology to quantify DNA using Sanger sequencing, that we validated experimentally with specially-engineered plasmids both in vitro and in co-transformed E. coli by and assessing our predictions with qPCR and fluorescence quantifications. The method relies on the alignment of the electropherograms from the query and reference samples, where we quantify the DNA concentration from the amplitude ratio of aligned electropherogram peaks. Our DNA quantification will allow quantifying genetic variants, including single-base natural polymorphisms or de novo mutations, from mixed Sanger sequencing reads, with consistent reduction of costs compared to canonical approaches such as qPCR.

synthetic biology↗

bioRxiv2022

Engineered gene circuits with reinforcement learning allow bacteria to master gameplaying

Gene circuits enable cells to make decisions by controlling the expression of genes in reaction to specific environmental factors1. These circuits can be designed to encode logical operations2-7, but implementation of more complex algorithms has proved more challenging. Directed evolution optimizes gene circuits8 without the need for design knowledge9, but adjusting multiple genes and conditions10 in genotype searches poses challenges11. Here we show a multicellular sensor system, AdaptoCells, in Escherichia coli, that can evolve complex behavior through an accelerated adaptation to chemical environments. AdaptoCells recognize chemical patterns and act as a decision-making system. Using an iterative improvement method, we demonstrate that the AdaptoCells can evolve to achieve mastery in the game of tic-tac-toe, demonstrating an unprecedented level of complexity for engineered living cells. We provide an effective and straightforward way to encode complexity in gene circuits, allowing for fast adaptation in response to dynamic environments and leading to optimal decisions.

synthetic biology↗

bioRxiv2021

Resistance to a CRISPR-based gene drive at an evolutionarily conserved site is revealed by mimicking genotype fixation

CRISPR-based homing gene drives can be designed to disrupt essential genes whilst biasing their own inheritance, leading to suppression of mosquito populations in the laboratory. This class of gene drives relies on CRISPR-Cas9 cleavage of a target sequence and copying ( homing) therein of the gene drive element from the homologous chromosome. However, target site mutations that are resistant to cleavage yet maintain the function of the essential gene are expected to be strongly selected for. Targeting functionally constrained regions where mutations are not easily tolerated should lower the probability of resistance. Evolutionary conservation at the sequence level is often a reliable indicator of functional constraint, though the actual level of underlying constraint between one conserved sequence and another can vary widely. Here we generated a novel gene drive in the malaria vector Anopheles gambiae, targeting an ultra-conserved target site in a haplosufficient essential gene (AGAP029113) required during mosquito development, which fulfils many of the criteria for the target of a population suppression gene drive. We then designed a selection regime to experimentally assess the likelihood of generation and subsequent selection of gene drive resistant mutations at its target site. We simulated, in a caged population, a scenario where the gene drive was approaching fixation, where selection for resistance is expected to be strongest. Continuous sampling of the target locus revealed that a single, restorative, in-frame nucleotide substitution was selected. Our findings show that ultra-conservation alone need not be predictive of a site that is refractory to target site resistance. Our strategy to evaluate resistance in vivo could help to validate candidate gene drive targets for their resilience to resistance and help to improve predictions of the invasion dynamics of gene drives in field populations. Author summaryGene drives have the potential to be applied as novel control strategy of disease-transmitting mosquitoes, by spreading genetic traits that suppress or modify the target population. Many gene drive elements work by recognising and cutting a specific target sequence in the mosquito genome and copying themselves into that target sequence allowing the gene drive to increase in frequency in the population. Like other mosquito control interventions, efficacy will greatly depend on minimising the development of resistance to the gene drive mechanism - most likely via a change in the target sequence that prevents further cutting. One strategy to reduce resistance is to target sequences that are highly conserved, which implies that changes cannot easily be tolerated. We developed a strategy that simulates high selection pressure, under which resistance is most likely to emerge, and therefore provides a stringent test of its propensity to arise. Unlike previous results with another gene drive, we recovered a resistant allele within a few generations of gene drive exposure and at high frequency. Our results show that conserved sequences can vary hugely in ability to tolerate mutations and highlights the need to functionally validate future candidate gene drive target sites for their robustness to resistance.

genetics↗

bioRxiv2019

The potential for a released autosomal X-shredder becoming a driving-Y chromosome and invasively suppressing wild populations of malaria mosquitoes

Synthetic sex-ratio distorters based on X-chromosome shredding are predicted to be more efficient than sterile males for population suppression of malaria mosquitoes using genetic control. X-chromosome shredding operates through the targeted elimination of X-chromosome-bearing gametes during male spermatogenesis, resulting in males that have a high fraction of male offspring. Strains harboring autosomal constructs containing a modified endonuclease I-PpoI have now been developed in the malaria mosquito Anopheles gambiae, resulting in strong sex-ratio distortion towards males. Data are being gathered for these strains for submission of regulatory dossiers for contained use and subsequent field release in West Africa. Since autosomal X-shredders are transmitted in a Mendelian fashion and can be selected against their frequency in the population is expected to decline once releases are halted. However, any unintended transfer of the X-shredder to the Y-chromosome could theoretically change these dynamics: This could lead to 100% transmission of the newly Y-linked X-shredder to the predominant male-biased offspring and its insulation from negative selection in females, resulting in its potential spread in the population and ultimately to suppression. Here, we analyze plausible mechanisms whereby an autosomal X-shredder could become linked to the Y-chromosome after release and provide data regarding its potential for activity should it become linked to the Y-chromosome. Our results strongly suggest that Y-chromosome linkage through remobilization of the transposon used for the initial genetic transformation is unlikely, and that, in the unexpected event that the X-shredder becomes linked to the Y-chromosome, expression and activity of the X-shredder would likely be inhibited by meiotic sex chromosome inactivation. We conclude that a functioning X-shredding-based Y-drive resulting from a naturally induced transposition or translocation of the transgene onto the Y-chromosome is unlikely.

genetics↗

bioRxiv2018

Improved CRISPR-based suppression gene drives mitigate resistance and impose a large reproductive load on laboratory-contained mosquito populations

CRISPR-Cas9 nuclease-based gene drives rely on inducing chromosomal breaks in the germline that are repaired in ways that lead to a biased inheritance of the drive. Gene drives designed to impair female fertility can suppress populations of the mosquito vector of malaria. However, strong unintended fitness costs, due to ectopic nuclease expression, and high levels of resistant mutations, limited the potential of the first generation of gene drives to spread. Here we show that changes to regulatory sequences in the drive element, designed to contain nuclease expression to the germline, confer improved fecundity over previous versions and generate drastically lower rates of target site resistance. We employed a genetic screen to show that this effect is explained by reduced rates of end-joining repair of DNA breaks at the target site caused by deposited nuclease in the embryo. Highlighting the impact of deposited Cas9, many of the mutations arising from this source of nuclease activity in the embryo are heritable, thereby having the potential to generate resistant target sites that reduce the penetrance of the gene drive. Finally, in cage invasion experiments these gene drives show improved invasion dynamics compared to first generation drives, resulting in greater than 90% suppression of the reproductive output and a delay in the emergence of target site resistance, even at a resistance-prone target sequence. We shed light on the dynamics of generation and selection of resistant alleles in a population by tracking, longitudinally, the frequency of resistant alleles in the face of an invading gene drive. Our results illustrate important considerations for future gene drive design and should expedite the development of gene drives robust to resistance.

genetics↗

bioRxiv2017

Cross-species Y chromosome function between malaria vectors of the Anopheles gambiae species complex

Y chromosome function, structure and evolution is poorly understood in many species including the Anopheles genus of mosquitoes, an emerging model system for studying speciation that also represents the major vectors of malaria. While the Anopheline Y had previously been implicated in male mating behavior, recent data from the Anopheles gambiae complex suggests that, apart from the putative primary sex-determiner, no other genes are conserved on the Y. Studying the functional basis of the evolutionary divergence of the Y chromosome in the gambiae complex is complicated by complete F1 male hybrid sterility. Here we used an F1xF0 crossing scheme to overcome a severe bottleneck of male hybrid incompatibilities and enabled us to experimentally purify a genetically labelled A. gambiae Y chromosome in an A. arabiensis background. Whole genome sequencing confirmed that the A. gambiae Y retained its original sequence content in the A. arabiensis genomic background. In contrast to comparable experiments in Drosophila, we find that the presence of a heterospecific Y chromosome has no significant effect on the expression of A. arabiensis genes and transcriptional differences can be explained almost exclusively as a direct consequence of transcripts arising from sequence elements present on the A. gambiae Y chromosome itself. We find that Y hybrids show no obvious fertility defects and no substantial reduction in male competitiveness. Our results demonstrate that, despite their radically different structure, Y chromosomes of these two species of the gambiae complex that diverged an estimated 1.85Myr ago function interchangeably, thus indicating that the Y chromosome does not harbor loci contributing to hybrid incompatibility. Therefore, Y chromosome gene flow between members of the gambiae complex is possible even at their current level of divergence. Importantly, this also suggests that malaria control interventions based on sex-distorting Y drive would be transferable, whether intentionally or contingent, between the major malaria vector species.

genetics↗

bioRxiv2017

The creation and selection of mutations resistant to a gene drive over multiple generations in the malaria mosquito

Gene drives have enormous potential for the control of insect populations of medical and agricultural relevance. By preferentially biasing their own inheritance gene drives can rapidly introduce genetic traits even if these confer a negative fitness on the population.\n\nWe have recently developed gene drives based on a CRISPR nuclease construct that is designed to disrupt key genes essential for female fertility in the malaria mosquito. The construct copies itself and the associated genetic disruption from one homologous chromosome to another during gamete formation, in a process called homing that ensures the majority of offspring inherit the drive. Such drives have the potential to cause long-lasting, sustainable population suppression though they are also expected to impose a large selection pressure for resistance in the mosquito. One of these population suppression gene drives showed rapid invasion of a caged population over 4 generations, establishing proof of principle for this technology. In order to assess the potential for the emergence of resistance to the gene drive in this population we allowed it to run for 25 generations and monitored the frequency of the gene drive over time. Following the initial increase of the gene drive we noticed a gradual decrease in its frequency that was accompanied emergence of small, nuclease-induced mutations at the target gene that are resistant to further cleavage and restore its functionality. Such mutations show rates of increase consistent with positive selection in the face of the gene drive. Our findings represent the first documented example of selection for resistance to a synthetic gene drive and lead to important design recommendations and considerations in order to mitigate for resistance for future gene drive applications.

evolutionary biology↗