In Bacteria and Archaea, mobile genetic elements (MGEs) are extremely diverse in terms of size, structure and mobilization mechanism, ranging from minimal non-autonomous sequences (<100 bp) to complex elements (>100,000 bp) capable of mobilizing many passenger genes. MGEs drive genome evolution and horizontal gene transfer (HGT), shaping the dissemination of functional traits across species, including antimicrobial resistance genes (ARGs) and virulence factors. Despite their importance, the distribution and transmission of MGEs in complex microbial communities remains understudied, largely due to the lack of methods for their systematic identification in metagenomic data and the limited scope of existing databases. In this thesis, I developed a comparative genomics framework to systematically identify insertions flanked by conserved regions in 1,345,857 prokaryotic genomes. This approach reconstructed 9,008,672 insertion clusters (ICs), capturing 61.4% of MGEs in public databases while revealing a vast diversity of previously uncharacterized elements. Integrating homology annotations, structural motif detection and machine learning predictions, I assigned 29.2% of ICs to known MGE classes, expanding their diversity by 32-fold. Functional analyses showed that ICs are enriched in canonical MGE-associated traits, such as ARGs, virulence factors, secondary metabolic pathways and prokaryotic defense systems. Mapping ICs across isolate genomes revealed extensive HGT across the prokaryotic tree of life, providing direct evidence of mobilization, and showed that 89.5% of horizontally transferred ICs were previously unknown. Together, these results represent the most comprehensive characterization of the prokaryotic mobilome to date and reveal a large diversity of uncharacterized elements, providing a resource for the exploration of MGEs at the metagenomic scale. In the second part of this thesis, I leverage large metagenomic data to explore the diversity of programmable nucleases for the development of new genome editing tools. CRISPR-Cas systems, which provide adaptive immunity against MGEs, have been widely repurposed for genome editing. However, clinical applications of currently available Cas nucleases remain limited by several factors, including activity, specificity, targeting requirements and efficient in vivo delivery. In particular, the widely used SpCas9 is not compatible with single adeno-associated viral (AAV) vector delivery, due 8 to its size, and is restricted to targets flanked by an NGG protospacer adjacent motif (PAM). To address these limitations, I developed a computational pipeline to identify and characterize TnpB proteins, compact programmable nucleases encoded by widespread MGE families (IS200/605 and IS607). By analyzing 330,895 TnpB orthologs from 14,127 species, I selected 25 candidates for experimental characterization. This led to the identification of ISPmu1, a TnpB from Pasteurella multocida that is active in human cells and represents a promising candidate for the development of compact genome editors. In parallel, I developed PAMpredict, a computational tool to accurately predict the PAM sequence of Cas9 nucleases. Applying this tool at scale revealed that natural PAM diversity across prokaryotes is sufficient to target almost all disease-causing mutations in the human genome with allele specificity. Overall, this thesis demonstrates that large metagenomic data enables the systematic exploration and characterization of MGEs and programmable nucleases across prokaryotes.
Matteo Ciciani· Institutional Research Infor...· 0 citations
Fusarium oxysporum is a significant threat to agriculture and One Health, requiring advanced molecular tools for functional genomic analyses and biological control agent development. Existing gene-editing methods are hampered by costly protoplast preparation protocols and by CRISPR-Cas9 limitations, such as restricted protospacer adjacent motif (PAM) sequences and complex guide RNA requirements. We engineered an efficient CRISPR/Cpf1 system that overcomes these issues through three main innovations: small-scale protoplast generation using filter column-based methods that greatly reduce enzyme consumption while simplifying workflows, a CRISPR/Cpf1 system with shorter guide RNA design and staggered DNA cleavage to promote homologous recombination, and minimal homology arm strategies that significantly decrease cloning complexity. Extensive validation confirms successful gene targeting with molecular verification and functional analysis via standardized pathogenicity assays. This integrated platform offers affordable, accessible tools for systematic F. oxysporum research, enhancing fundamental understanding of plant-pathogen interactions and supporting high-throughput screening vital for agricultural biotechnology and biological agent development.
Nucleic acid therapeutics, particularly DNA and messenger RNA (mRNA), provide programmable platforms for cancer vaccination, local expression of immunomodulatory proteins, and gene regulation. Their therapeutic performance, however, is strongly constrained by extracellular degradation, route-dependent biodistribution, inefficient cellular uptake, and limited delivery to antigen-presenting cells. Microneedle (MN) systems offer a minimally invasive means of depositing nucleic acids in the epidermis and dermis, where immune cells and lymphatic networks can support local antigen presentation and immune priming. This review critically examines MN-mediated DNA and mRNA delivery for cancer immunotherapy. We first compare the biological requirements and delivery barriers of DNA and mRNA, and then relate these requirements to MN architecture, material selection, mechanical performance, cargomatrix interactions, and release behavior. Representative DNA vaccine, gene-editing, mRNA vaccine, and combination-therapy platforms are discussed with emphasis on what each design solves and which limitations remain. Particular attention is given to delivered-dose variability, intracellular transfection, cargo stability during fabrication and storage, manufacturing consistency, safety after repeated application, and the restricted applicability of MNs to skin and accessible lesions. Rather than treating MNs as a universal replacement for systemic delivery, we position them as a route-specific platform whose clinical value will depend on rational cargodevice matching, standardized potency testing, and validation in translational models.
Haowei Liu, Qiong Yi, Ling Mei et al.· NANO· 0 citations
For over a century, the social and biological sciences have struggled with reductionist frameworks that divide the human condition into competing columns of nature versus nurture. This paper introduces a unified theoretical framework formalised by the conceptual equation A + B = 1, where A represents human biological inheritance and B represents dynamic cultural systems. By assigning a baseline value of 0.5 to both variables, this model establishes a rigorous guardrail against both biological determinism and radical cultural blank-slate models. This paper demonstrates that while the overarching importance of both systems remained balanced, their operational weights fluctuate dynamically depending on the trait analyzed—ranging from high biology physiological reflexes to high-culture social structures. Utilising classic anthropological case studies – including West African sickle-cell allele selection via yam cultivation and adult lactase persistence driven by pastoralism—we illustrate how cultural practices act as selective pressures that alter genetic frequencies. This paper also evaluates how modern biotechnologies, such as CRISPR gene editing and neural interfaces, allow culture to directly rewrite biological code, altering the temporal sync of human evolution. Finally, this framework provides a vital heuristic device for the natural and social sciences, including anthropology, provided that the human evolutionary trajectory is essentially a biocultural synthesis that cannot be solved without accounting for both halves of the equation.
R Khongsdier· International Journal on Sci...· 0 citations
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Abstract Gene editing technologies have evolved significantly over the past few decades, revolutionising biomedical research and therapeutics. Initially, restriction enzymes provided researchers with basic tools for deoxyribonucleic acid (DNA) manipulation. However, the advent of engineered nucleases, such as zinc finger nucleases (ZFNs) and transcription activator‐like effector nucleases (TALENs), marked significant advancements for more precise and customisable genome modifications. The discovery of CRISPR–Cas9 further transformed gene editing, offering a more efficient, cost‐effective and versatile approach. The success of CRISPR in transgenic mouse generation and clinical trials is a testament to this. However, challenges such as off‐target effects, immunogenicity and ethical concerns surrounding germline editing and eugenics remain. This review will discuss the discovery, mechanisms and applications of gene editing tools as they have evolved. It will also introduce modern iterations of gene editing to tackle off‐target effects and the ethical responsibility that comes with it. Key Concepts Gene editing has evolved from restriction enzymes to programmable nucleases capable of precise genome modifications. Zinc finger nucleases were the first major platform for targeted DNA cleavage but came with technical challenges. Transcription activator‐like effector nucleases (TALENs) improved gene editing through simpler and more predictable DNA sequence recognition. CRISPR–Cas9 transformed gene editing by using guide RNA to direct efficient, low‐cost and scalable DNA targeting. Off‐target mutations, delivery barriers and immune responses remain major technical challenges for therapeutic gene editing. Newer iterations of the CRISPR–Cas system offer higher fidelity gene editing and applications beyond DNA cleavage. Germline editing, mosaicism, eugenics and informed consent remain central ethical concerns in gene editing.
Oscar Sorrell, Maria Victoria Niklison-Chirou· Encyclopedia of Life Science...· 0 citations
Induced pluripotent stem cell-derived chimeric antigen receptor natural killer (iPSC-CAR-NK) cells are an evolving off-the-shelf cellular immunotherapy platform with potential for scalable manufacturing, product standardization, and multiplex engineering.This review summarizes recent progress in clinical-grade manufacturing, quality-attribute definition, and natural killer cell-adapted engineering strategies, including chimeric antigen receptor design, gene editing, tumor microenvironment adaptation, single-cell and multi-omics-guided optimization, and synthetic biologybased control.In addition, this review discusses recent progress in the application of iPSC-CAR-NK cells to hematologic malignancies and solid tumors and cautiously examines the early translational signal suggested by a single compassionate-use report in systemic sclerosis.To move beyond a descriptive listing of engineering strategies, we propose a multilevel analytical framework encompassing product definition, effector execution, tissue delivery, host interaction, and translational implementation.This framework is used to evaluate the functional roles, interrelationships, and limiting factors of engineering modules during clinical translation.Current evidence suggests manageable safety profiles and preliminary antitumor or immunomodulatory activity in some settings, but major barriers remain, including limited persistence, insufficient solidtumor infiltration, host immune clearance, antigen escape, multiplex-engineering risks, and immature quality-control and regulatory standards.
Xu-Lin Liu, Si-Min Han, Gang-Hui Ye et al.· World Journal of Stem Cells· 0 citations
Supporting data for the manuscript: Chaudhari et al., Synthesis cost is a hidden driver of convergent amino acid composition in plastid ribosomal proteins. List of contents:Data S1 Sequence alignments of all surveyed plastid and nuclear genes in Orobanchaceae, Geraniaceae, Fabaceae, and Caryophyllaceae. These genes included the plastid-encoded plastid ribosome genes (CpPRP), nuclear-encoded plastid-targeted ribosomal proteins (NuPRP), nuclear-encoded plastid-targeted non-ribosomal proteins (NuCpOT), and nuclear-encoded cytosolic ribosomal proteins (NuCyRP). The RNA editing sites of plastid genes were predicted using PREPACT v3 and masked using a custom Python script. The N-terminal peptides of nuclear-encoded plastid-targeted genes were predicted using TargetP v2.0 and trimmed. Data S2 Changes in protein folding stability estimated by ELASPIC2. Protein stability was measured by changes in free energy of protein folding (ΔΔG) using the deep learning powered tool ELASPIC. Pairwise comparisons were performed between 1) observed natural protein pairs (e.g., one Cymbarieae species and a closely related outgroup) and 2) reconstructed ancestral proteins with artificial mutations (e.g., reconstructed ancestral protein of Cymbarieae with introduced R→K mutations).
Arnav Chaudhari, Palash Sethi, Yvemirca Vilbrun et al.· Zenodo (CERN European Organi...· 0 citations
The Bacillus pumilus 3-19 strain exhibits increased secretion of various hydrolases, including proteases, and is a promising plant growth-promoting (PGP) agent. A unique secreted minor metalloproteinase, MprBp, which has no homologs among prokaryotic enzymes, has been identified in its genome. The role of the enzyme in B. pumilus cells is poorly understood. Using CRISPR/Cas9 genome editing, a deletion mutant of B. pumilus 3-19 with an inactivated mprBp gene was obtained. Comparative analysis of the mutant and native strains by quantitative reverse transcription PCR (RT-qPCR) revealed a significant decrease in the expression of other minor proteases. The mutant strain also demonstrated a two-fold increase in biofilm formation, suggesting that MprBp acts as a negative regulator of this process. Additionally, seed treatment with B. pumilus ΔmprBp led to an increase in the morphometric parameters of barley seedlings (Hordeum vulgare L.).
Damir I. Khasanov, Н. Л. Рудакова, Iuliia V. Danilova et al.· Microorganisms· 0 citations
Salinity stress affects rice productivity due to reduced growth and sodium ion toxicity. Previously, we identified a splice variant of RADIATION SENSITIVE23a (RAD23a) as the potential basis for variation in salt-tolerance in rice germplasm. RAD23 is a known moonlighting protein associated with protein degradation. To validate the role of RAD23a in salt stress response, we characterized gene edited mutant lines that targeted the UBL and UBA2 domains of this protein. Mutation in either domain promoted shoot growth under saline and control conditions. The mutants also differed from wildtype plants in Na and K accumulation in roots and shoots under salt stress. Transcriptome analysis of mutants versus wildtype showed differential transcript abundance of multiple inorganic phosphate (Pi) starvation related genes, including OsSPX2 and OsPHO2. As a result, mutants accumulate higher Pi compared to wildtype plants. The two allelic groups for RAD23a locus also differ in root and shoot phosphorus (P) content. Further, we show that RAD23a interacts with OsSPX2, a negative post-translational regulator of OsPHR2, the master regulator of Pi starvation response. Mutants have higher shoot growth and Pi levels under low Pi conditions, linking enhanced growth of mutants to increased Pi uptake. The UBA2 domain specific mutants have higher single grain weight and per plant grain weight than wildtype. In summary, we show that the RAD23a regulates differential growth, salt response and Pi uptake in rice in a domain-specific manner supporting the moonlighting roles of RAD23a in salt tolerance and phosphorus-dependent shoot growth.
Shohei Oguro, Usama Ahmad, Anil Kumar Nalini Chandran et al.· bioRxiv (Cold Spring Harbor...· 0 citations
Drought stress poses a major threat to global wheat (Triticum aestivum L.) productivity by impairing physiological processes and inducing oxidative damage. Mutation breeding provides a valuable approach to generate novel genetic variation and identify stress-tolerant germplasms. In this study, phenotypic, physiological, and transcriptomic analyses were integrated to elucidate the drought adaptation mechanisms of a gamma-ray-induced mutant wheat line, PL6, alongside its wild-type parent, PL1. Under osmotic stress and soil drought conditions, PL6 exhibited an enhanced germination rate and higher photosynthetic efficiency (Fv/Fm). Furthermore, PL6 maintained lower malondialdehyde (MDA) accumulation, which was supported by elevated activities of antioxidant enzymes including SOD, APX, and CAT. Time-series transcriptomic analysis via WGCNA and GSEA revealed that PL6 actively maintains environmental sensing, transmembrane transport, and photosynthetic processes under PEG-induced osmotic stress. Conversely, pathways associated with the cell cycle and DNA metabolism were transiently suppressed. To isolate key regulatory genes without computational bias, a multi-algorithm machine learning framework—combining Random Forest, LightGBM, and LASSO—was applied to variance-stabilizing transformed (VST) expression profiles. This approach successfully identified 45 consensus core drought-responsive genes enriched in targeted protein turnover, redox balance, cell wall restructuring, and lipid metabolism, from which ten representative candidate genes were experimentally validated via qRT-PCR. Collectively, this study demonstrates an effective analytical framework for selection of high-confidence transcripts, providing candidate targets for future targeted gene editing and molecular breeding in wheat.
Min Jeong Hong, Ryu Jeong Kim, So Jin Park et al.· Agriculture· 0 citations
Supporting data for the manuscript: Chaudhari et al., Synthesis cost is a hidden driver of convergent amino acid composition in plastid ribosomal proteins. List of contents:Data S1 Sequence alignments of all surveyed plastid and nuclear genes in Orobanchaceae, Geraniaceae, Fabaceae, and Caryophyllaceae. These genes included the plastid-encoded plastid ribosome genes (CpPRP), nuclear-encoded plastid-targeted ribosomal proteins (NuPRP), nuclear-encoded plastid-targeted non-ribosomal proteins (NuCpOT), and nuclear-encoded cytosolic ribosomal proteins (NuCyRP). The RNA editing sites of plastid genes were predicted using PREPACT v3 and masked using a custom Python script. The N-terminal peptides of nuclear-encoded plastid-targeted genes were predicted using TargetP v2.0 and trimmed. Data S2 Changes in protein folding stability estimated by ELASPIC2. Protein stability was measured by changes in free energy of protein folding (ΔΔG) using the deep learning powered tool ELASPIC. Pairwise comparisons were performed between 1) observed natural protein pairs (e.g., one Cymbarieae species and a closely related outgroup) and 2) reconstructed ancestral proteins with artificial mutations (e.g., reconstructed ancestral protein of Cymbarieae with introduced R→K mutations).
Arnav Chaudhari, Palash Sethi, Yvemirca Vilbrun et al.· Zenodo (CERN European Organi...· 0 citations
Genetic Disorders: Molecular Mechanisms, Diagnostic Evolution, Precision Therapeutics and Future Directions Description Genetic Disorders: Molecular Mechanisms, Diagnostic Evolution, Precision Therapeutics and Future Directions is a comprehensive narrative review examining the current understanding and rapidly evolving landscape of genetic disorders. The review integrates advances in molecular genetics, genomic diagnostics, precision medicine, and emerging therapeutic strategies to provide a broad perspective on the diagnosis and management of genetic diseases. The review explores the molecular mechanisms underlying genetic disorders, including pathogenic genetic variants, mutations, genomic alterations, gene regulation, and disrupted molecular pathways involved in disease development and progression. It further examines the evolution of genetic diagnosis from conventional approaches toward advanced genomic technologies, including next-generation sequencing (NGS), whole-exome sequencing (WES), whole-genome sequencing (WGS), and molecular diagnostic approaches. A major focus of this review is the development of precision therapeutics and personalized medicine. Emerging approaches such as gene therapy, genome editing, RNA-based therapies, targeted molecular treatments, and genotype-guided therapeutic strategies are discussed in the context of their potential to transform the management of genetic disorders. The review also addresses important challenges in genomic medicine, including variant interpretation, genetic counseling, ethical considerations, accessibility of advanced diagnostic technologies, clinical implementation, and the integration of genomic data into routine healthcare. Finally, the article highlights future directions in genetic medicine, including the potential contributions of artificial intelligence, bioinformatics, multi-omics technologies, genomic data analysis, and precision healthcare. These developments may facilitate earlier diagnosis, improved disease classification, individualized treatment selection, and better clinical outcomes. Overall, this narrative review provides an integrated overview of the transition from traditional genetic diagnosis and management toward genomics-driven, mechanism-based, and precision therapeutic approaches, while identifying key opportunities and challenges for future research and clinical practice. Keywords:Genetic Disorders; Molecular Genetics; Genetic Mutations; Genomic Medicine; Molecular Mechanisms; Genetic Diagnosis; Next-Generation Sequencing; Whole-Exome Sequencing; Whole-Genome Sequencing; Precision Medicine; Precision Therapeutics; Gene Therapy; Genome Editing; RNA-Based Therapy; Personalized Medicine; Bioinformatics; Artificial Intelligence; Genomic Technologies.
Dr.Zeeshan Qaiser· Zenodo (CERN European Organi...· 0 citations
A new method for surgically removing training examples from a model reveals that as datasets grow, the link between what a model learns and what it produces dissolves.
MIT News · Artificial Intelligence· news.mit.eduAug 17, 2026
