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At least 19 recordsLinked to original sources
PubMed2025

Genetic Engineering and Screening Using Base Editing and Inducible Gene Knockout.

Genetic engineering and screening in human cells are powerful techniques for the precise and comprehensive identification and analysis of gene and protein domain functions. Genome-wide knockout screens have been extensively utilized to discover essential genes, tumor suppressors, and genes that regulate responses to various chemicals, including antimitotic and therapeutic drugs. The advent of base editors, which facilitate the targeted mutation of single amino acids, has advanced the identification of critical and functional domains or motifs. In this context, we outline methods for creating efficient base editor and inducible knockout cell lines for targeted gene manipulation and conducting genetic screens to elucidate the roles of genes and their domains within a specific cell biological context.

Humans↗
PubMed1976

[Successes and prospects for genetic engineering].

The review of literature (1970-1976) on problems of gene engineering is given. Gene engineering is pointed out to be a new method of modern biology and a new page of modern molecular genetics. Gene engineering detected a real possibility of artificial creating living hybrid organisms, i.e. constructing functional recombinant DNA molecules according to a project of investigator, but not to possibilities of crossing. The determination of gene engineering (in contrast with genetical engineering) is given in the first division of the article. Genetical engineering is a construction of hybrid organisms on the basis of recombination between non-homologous chromosomes cy crossing. Genetical engineering is based on sex crossing, thus the application of this method is restricted by crossability (i.e. experiments in vivo), which possibilities are determined by taxonomical limits. Gene engineering is a new method of operating directly with genes. It permits constructing in vitro any hybrid genomes desirable. There is no limits of combining ability for gene engineering. Three main stages of constructing hybrid genomes should be taken into account for the proper determination of gene engineering as a method of genome constructing: 1) the gene isolation; 2) their cross-linking in vitro; 3) the transfer of hybrid DNA into recipient cell or its genome. The cardinal stage of gene engineering is the construction of hybrid DNA, cross-linking any initial DNAs from any remote animals, plants and bacteria. All the methods known of gene isolation are described. The chemical method of gene isolation is based on that case, when DNA of some gene differs in its physico-chemical characteristics from total DNA, for example, DNAs of genes coding ribosomal RNAs or sea urchine histone DNA. Isolation of promotors and operators using DNA dependent RNA polymerase, which recognizes promotors, repressor and operator DNA, should also be considered as the chemical method of gene isolation. Restrictase method, which is also well known, is convenuent because the restricts have long enough sticky ends, which is important for the following gene cross-linking. The method of total restriction, reported by Lederberg et al. and Debabov et al., is described. The phage method (in particular, Shimada method) is given, permitting the direct integration of lambda phage into a number of sites of Escherichia coli chromosome. Gene engineering method of gene isolation is mentioned, in particular, the data of Kameron et al. on hybrid phages carrying DNA ligase gene, and Clark a. Carbon on hybrid plasmids carrying triptophane and arabinose operons genes. These methods are called "shot gun". Methods of gene isolation from higher organisms are less developed. A method of gene isolation using so called colony hybridization (according to Grünstein and Hognes) is also given...

Bacteriophages↗
PubMed2025

A comprehensive review of genomic-scale genetic engineering as a strategy to improve bacterial productivity.

Bacterial genome engineering has evolved to provide increasingly precise, robust and rapid tools, driving the development and optimization of bacterial production of numerous compounds. The field has progressed from early random mutagenesis methods, labour-intensive and inefficient, to rational and multiplexed strategies enabled by advances in genomics and synthetic biology. Among these tools, CRISPR/Cas has stood out for its versatility and its ability to achieve precision levels ranging from 50% to 90%, compared to the 10-40% obtained with earlier techniques, thereby enabling remarkable improvements in bacterial productivity. Nevertheless, like its predecessors, it still demands continuous refinement to reach full maturity. In this context, the present review addresses the lack of a unified overview by summarizing historical milestones and practical applications of genomic engineering tools in bacteria. It integrates diverse approaches to provide a comprehensive perspective on the evolution and prospects of these fundamental biotechnological tools.

Bacteria↗
PubMed1979

Gentic engineering for practical application.

Genetic engineering has ushered in a new era in biology. Although many problems are still to be solved, there are examples that point to a possible later application for the benefit of mankind: Bacteria can be manipulated to degrade crude-oil spillages, to produce human insulin and to bind nitrogen from the air. If all the bacteria that are indigenous to agricultural soils could be made to bind nitrogen, an increase in soil fertility might well result.

Arthrobacter↗
PubMed2025

Boolean matrix logic programming for active learning of gene functions in genome-scale metabolic network models.

Reasoning about hypotheses and updating knowledge through empirical observations are central to scientific discovery. In this work, we applied logic-based machine learning methods to drive biological discovery by guiding experimentation. Genome-scale metabolic network models (GEMs) - comprehensive representations of metabolic genes and reactions - are widely used to evaluate genetic engineering of biological systems. However, GEMs often fail to accurately predict the behaviour of genetically engineered cells, primarily due to incomplete annotations of gene interactions. The task of learning the intricate genetic interactions within GEMs presents computational and empirical challenges. To efficiently predict using GEM, we describe a novel approach called Boolean Matrix Logic Programming (BMLP) by leveraging Boolean matrices to evaluate large logic programs. We developed a new system, [Formula: see text], which guides cost-effective experimentation and uses interpretable logic programs to encode a state-of-the-art GEM of a model bacterial organism. Notably, [Formula: see text] successfully learned the interaction between a gene pair with fewer training examples than random experimentation, overcoming the increase in experimental design space. [Formula: see text] enables rapid optimisation of metabolic models to reliably engineer biological systems for producing useful compounds. It offers a realistic approach to creating a self-driving lab for biological discovery, which would then facilitate microbial engineering for practical applications.

Active learning↗
PubMed2026

Prophage landscapes in clinical MRSA: safety profiling and discovery of Lys81, a broad-spectrum bacteriolytic enzyme.

INTRODUCTION: Methicillin-resistant Staphylococcus aureus (MRSA) poses a significant threat to global healthcare, requiring novel therapeutic strategies. Prophages, latent phage genomes integrated into bacterial chromosomes, are important resources for antimicrobial development due to their genomic stability and genetic engineering potential. METHODS: In this study, we performed genomewide sequencing on 329 MRSA isolates to predict prophage sequences, followed by analyses of these prophages-including examinations of virulence genes, antibiotic resistance genes, homologous proteins of pathogenic MRSA phages, and functional predictions of these homologous proteins-to evaluate their safety and value as genetic engineering scaffolds and to screen for novel broadspectrum bacteriolytic enzymes. RESULTS: Our data indicate that 85.7% (282/329) of strains carried complete prophage sequences; 64 strains lacked virulence factors or genes, meeting the core criteria for safe vectors. Resistance screening found only 6 prophages carried msrA, confirming the biosafety of the remaining strains. A significant correlation existed between prophage virulence gene capacity and genomic structure (R2 = 0.99986684, p = 3.64e-69). High-virulence clusters (>10 factors) showed high structural similarity; 10 characteristic sequences linked to S. aureus phages and their prevalence patterns were identified via conserved motif analysis. Collinearity analysis with reference to virulent MRSA phages and 3D structural predictions of orthologous proteins identified two lysozymes and a host-recognition device. Notably, Lys81, an N-acetylmuramoyl-L-alanine amidase ortholog, was prioritized and characterized as a broad-spectrum lytic enzyme. Our data show Lys81 has key properties: (1) Broad-spectrum antibacterial activity, lysing 52.3% (23/44) of clinical S. aureus strains and cross-acting against Gram-positive bacteria such as Pseudomonas aeruginosa and Listeria; (2) Excellent environmental adaptability, maintaining activity at pH 5.0 and 0°C, with 25 mM Na+ and Ca2 + enhancing function; (3) Potent biofilm clearance, achieving 83% MRSA biofilm reduction at 50 μg/mL; and (4) Favorable in vivo safety/efficacy, eradicating MRSA infections in lung organoid models with minimal cytotoxicity. DISCUSSION: This study establishes a theoretical foundation for the clinical translation of MRSA prophages, positioning Lys81 as a novel candidate for treating drug-resistant bacterial infections.

Lys81↗
PubMed2026

Proteomic Identification of Pig Xenoantigens for Clinical Xenotransplantation.

Xenotransplantation using genetically engineered pig organs offers a promising solution to the shortage of donor organs for life-saving transplantations. However, human-preformed antibodies against unknown pig xenoantigens remain a significant barrier to successful xenotransplantation. Current methods for characterizing these antibodies or xenoantigens are limited to cellular-level cross-match assays. In this study, we developed a novel approach to identify pig xenoantigens, including peptide and glycopeptide epitopes, that react with human-preformed antibodies. First, human-preformed antibodies against xenoantigens were enriched from plasma using immobilized pig kidney proteins. The enriched antibodies were then immobilized and used to isolate pig kidney proteins, peptides, and intact glycopeptides, followed by liquid chromatography-tandem mass spectrometry analysis. This dual-level approach identified 221 peptides corresponding to 153 proteins, with a significant enrichment of plasma membrane and extracellular proteins. Notably, 11 peptides were unique to pig sequences, suggesting their potential role in driving xenogeneic immune responses. Glycoproteomic analysis identified 122 intact glycopeptides, predominantly complex/hybrid glycoforms, and Neu5Gc-containing glycans. Our method effectively identifies peptides and intact glycopeptides reactive to human-preformed antibodies, providing critical insights for discovering xenoantigens. These findings could guide genetic engineering strategies and enhance recipient candidate screening for xenotransplantation, ultimately increasing the feasibility and success of xenogeneic organ transplantation.

Animals↗
PubMed1975

[Present problems of somatic cell genetics].

A review of recent advances in genetics of somatic cells is given in the article. It contains three sections: 1) hybridization of somatic cells and its application to the mapping of genes and to the study of gene action; 2) relation of somatic cell genetics to genetic engineerings; 3) progress and perspectives of higher plant somatic cell genetics.

Animals↗
PubMed2026

Nonviral transposon‑engineered stem cells characterization: dose‑dependency between vector copy number and transgene expression.

Genetically engineered stem cells hold substantial promises for advancing regenerative medicine, yet ensuring their genomic safety remains a critical challenge. A key safety concern is vector copy number (VCN), which defines the number of integrated transgene copies per genome. Although ddPCR is used to assess VCN in virally transduced cells, its application in transposon‑engineered systems is limited. In this study, we extended VCN determination to non‑viral, transposon‑engineered stem cells. In alignment with FDA recommendations, the primary objective was to establish a robust and quantitative framework for interim VCN determination at the time of lot release. Specifically, we demonstrate that reliable interim VCN estimates increase in a dose‑dependent manner with increasing plasmid input. In addition, strong linear correlations between VCN and both EGFP median fluorescence intensity (MFI) and gene‑of‑interest (GOI) protein expression validate the accuracy of this framework. Furthermore, comparison of two distinct GOIs revealed gene‑specific differences in expression efficiency. Together, these findings validate a standardized VCN determination workflow that quantitatively links plasmid dose, genomic integration, and functional transgene expression. This workflow provides a systematic characterization of engineered cells, offering comprehensive information to support downstream risk‑based analyses to ensure the genomic safety and stability of the final cell product.

Transgenes↗
PubMed1977

[Conduct of Bacillus subtilis transformation in mice under conditions of immunologic suppression of exogenous DNAase 1 activity].

Bacillus subtilis transformation was conducted in the abdominal cavity of mice. The frequency of transformation was considerable decreased when bovine DNA-ase 1 (3-- 5microgram) was injected intraperitoneally to these animals. Immune rabbit gamma-globulins containing antibodies to bovine DNA-ase 1 inhibited in vivo the activity of DNA-ase 1, protected transforming DNA from the hydrolyzing effect of this enzyme. This model can be utilized in search for ways to preserve DNA injected into the animal organism for the purpose of genetical engineering.

Animals↗
PubMed2025

Efficient CRISPR/Cas9-mediated genome editing of phytoene desaturase in Musa-AAA: a critical step for genetic improvement of east African highland bananas.

East African highland bananas (EAHBs), locally referred to as "matooke", are an important staple crop in Uganda. The EAHBs have a triploid genome (AAA) with a large phenotypic diversity in the Great Lakes region of Africa and are challenged by both abiotic and biotic factors. The EAHBs have been improved through conventional breeding and genetic engineering though facing challenges such as genetic drag of unfavorable traits and complex regulatory processes, respectively. Therefore, a more precise approach for crop improvement such as genome editing is highly recommended. In the current study, we assessed the feasibility and applicability of the CRISPR/Cas9 mediated-genome editing in EAHBs. Two sgRNAs were designed from the Nakitembe phytoene desaturase (PDS) gene and used to edit the PDS gene in Nakitembe (NKT) and NAROBan5 (M30) cultivars. A total of 47 NKT and 130 M30 events were regenerated via agrobacterium-mediated transformation of banana embryogenic cell suspensions. Up to 100% and 94.6% albinism rates were observed in Nakitembe and M30 cultivars respectively with additional albino-variegated and variegated phenotypes observed in M30 only. Carotenoid analysis revealed a significant reduction of total carotenoid content in edited events with all complete albinos showing no detectable carotenoids implying that the carotenoid biosynthetic pathway was effectively disrupted. Sequence analysis revealed that all of the edited events had frameshift mutations leading to PDS disruption. Overall, this study presents the first report of CRISPR/Cas9 genome editing in EAHBs and more interestingly on a hybrid, M30 showing high precision and efficiency. This validated genome editing system provides a robust platform for targeted EAHB improvement.

CRISPR/Cas9↗
PubMed2026

Gene overexpression reduces inhibitory metabolites to enhance CHO cell growth and IgG1 production.

Controlling the generation of toxic by-products in mammalian bioprocess to maximize therapeutic protein production and glycosylation patterns is a challenge. Intracellular metabolism is often not well-regulated and known to secrete toxic intermediate by-products which hampers cellular performance and negatively impacts critical quality attributes (CQA) of cells. Previous studies have identified trigonelline (TRI), n-acetyl putrescine (NAP), aconitic acid (AA), and cytidine monophosphate (CMP) generated through CHO cell metabolism and verified their negative impacts on growth and antibody production. In this approach, a genetic engineering strategy was developed to control downstream accumulation of inhibitory metabolites. The study successfully identified four different metabolic genes in CHO cells, including Cat (nicotinate and nicotinamide metabolism) to control the generation of TRI, Got1 and Hoga1 (proline metabolism) to control the generation of NAP, Got1 (TCA cycle) to control the generation of AA, and Slc35a1 (n-glycan biosynthesis) to control the generation of CMP. Each target gene-of-interest (GOI) was cloned from CHO genomic library, inserted into linearized vector plasmid, and subsequently transfected into cells. CQA of the bioprocess realized 22-30% increase in peak cell density, 16-22% increase overall IVCD, with an improving growth rate during cellular expansion phase when comparing engineered cells against control cells. The study also conducted a follow-up quadruple transfection study where all four GOIs were co-transfected into cells at ¼ of the total DNA concentration per GOI. An increase in cellular performance was also realized, as increases in peak VCD (17% increase), cumulative IVCD (17% increase), and growth rate were achieved. Both studies also found higher IgG1 antibody synthesis when cell metabolism was better regulated, as the studies measured 4% to 40% titer increase across all engineered cells when compared against control cells. The study also measured higher levels of G1F and G2F glycans with decreased level of G0F across all transfected cells, further indicating improvement in bioprocess, as cells were able to produce a higher fraction of semi-complex and complex versus simple glycoforms. Further investigation revealed that Cat and Slc35a1 exhibited comparable expression levels in the MG condition to their single-gene conditions (within 1% and 10% difference, respectively), corresponding to modest titer improvements closest to the control. These findings suggest that when all four genes are co-expressed, Cat and Got1 may act as rate-limiting factors influencing both cellular phenotypes and titer production. In both studies, the concentrations of downstream metabolic inhibitors were measured to be significantly decreased when comparing engineered cells against control cells, further demonstrating that overexpression of genes to re-allocate metabolic fluxes away from synthesizing toxic by-products can significantly improve cellular growth and protein synthesis.

Animals↗
PubMed2024

Engineering a probiotic Bacillus subtilis for acetaldehyde removal: A hag locus integration to robustly express acetaldehyde dehydrogenase.

We have addressed critical challenges in probiotic design to develop a commercially viable bacterial strain capable of removing the intestinal toxin, acetaldehyde. In this study, we report the engineering of the hag locus, a σD-dependent flagellin expression site, as a stable location for robust enzyme production. We demonstrate constitutive gene expression in relevant conditions driven by the endogenous hag promoter, following a deletion of the gene encoding a post-translational regulator of σD, FlgM, and a point mutation to abrogate the binding of the translational inhibitor CsrA. Reporter constructs demonstrate activity at the hag locus after germination, with a steady increase in heterologous expression throughout outgrowth and vegetative growth. To evaluate the chassis as a spore-based probiotic solution, we identified the physiologically relevant ethanol metabolic pathway and the subsequent accumulation of gut-derived acetaldehyde following alcohol consumption. We integrated a Cupriavidus necator aldehyde dehydrogenase gene (acoD) into the hag locus under the control of the flagellin promoter and observed a rapid reduction in acetaldehyde levels in gut-simulated conditions post-germination. This work demonstrates a promising approach for the development of genetically engineered spore-based probiotics.

Acetaldehyde↗