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At least 19 recordsLinked to original sources
PubMed2026

Establishment of a CRISPR-Cas9 Library for Indica Rice and Identification of OsOPR5 (LOC_Os06g11210) as a Regulator of Root Architecture.

Functional characterization of a large number of rice genes remains a major challenge despite the availability of genome sequences and large-scale transcriptomic datasets. CRISPR-Cas9 library is a powerful approach for high-throughput targeted mutagenesis; however, its application in indica rice cultivars remains limited due to low transformation and regeneration efficiencies. In this study, we developed a CRISPR-Cas9 library targeting 12,000 rice genes and evaluated its utility for functional genomics in the indica cultivar MTU-1010. Sanger sequencing and NGS analysis of the plasmid library revealed high sgRNA coverage and more than 80% accuracy. Transformation of the developed library into the indica cultivar MTU-1010 resulted in a high target editing efficiency, with 90% of analyzed transgenic plants carrying mutations at the intended target site. Functional analysis of one homozygous mutant identified a previously uncharacterized role for OsOPR5 (LOC_Os06g11210), a member of the 12-oxophytodienoate reductase family in root architecture. The opr5 mutants exhibited significant reductions in lateral root number, seminal and crown root number, and root length, demonstrating that OsOPR5 positively regulates root system architecture in rice. Notably, endogenous jasmonic acid (JA) and JA-isoleucine levels were not significantly altered in the mutant, suggesting potential functional specialization or redundancy among rice OPR family members for JA accumulation. The root system architecture is a key determinant of water and nutrient acquisition; our results suggest that OsOPR5 may play an important role in adaptation under adverse environmental conditions. Collectively, this study establishes an efficient genome-editing platform for indica rice and identifies OsOPR5 as a novel regulator of root development.

Oryza↗
PubMed2026

Identification of novel inhibitors of Mycobacterium smegmatis growth through genome-wide overexpression of Cluster P3 mycobacteriophage Xavia genes.

Bacteriophages encode numerous genes with no known function, many of which can affect essential cellular processes when expressed in the bacterial host. For mycobacteriophages, genome-wide overexpression in Mycobacterium smegmatis can be used to identify proteins that impair growth. To evaluate the cytotoxic potential of the Cluster P3 phage Xavia, we constructed a plasmid library containing 71 predicted Xavia genes under the anhydrotetracycline inducible promoter pTet and screened this library in a plate-based cytotoxicity assay to measure impacts on M. smegmatis growth. Two genes prevented transformants recovery, consistent with toxicity under basal promoter leakiness, and inducible expression of 18 additional genes impaired growth. These inhibitory proteins include structural components; factors involved in DNA metabolism, lysogeny, and lysis; and several proteins with no known function. These results extend functional screening into a lineage of actinobacteriophages that has not previously been characterized, and identify new proteins that warrant further mechanistic analysis.

Mycobacterium smegmatis↗
PubMed2025

Experimental strategy for characterization of novel TnpB orthologs.

TnpB proteins encoded in IS200/IS605 and IS607 mobile genetic elements are among the most widespread proteins in the microbial world. They function as RNA-guided DNA nucleases that play a critical role in transposon proliferation and are the predecessors of CRISPR-Cas12 effector proteins of the type V CRISPR-Cas family. Small size of TnpB nucleases makes them an attractive alternative for larger Cas9 and Cas12 proteins in genome editing applications. However, only a small fraction of TnpB nucleases characterized to date are active in human cells, highlighting the need to identify new TnpB variants that can function as genome editors. Here, we present an experimental pipeline for the characterization of TnpB proteins by combining in silico analysis with in vitro assays. To validate it we determined guide RNA and identified TAM for a set of TnpB orthologs. The proposed workflow can be employed for rapid screening and characterization of the huge TnpB protein family to identify novel TnpB variants that might expand the genome editing toolbox.

Humans↗
PubMed2026

Host-aware Identification of Intrinsic Gene Expression Biopart Parameters using Combinatorial Libraries.

Model-based design in synthetic biology is limited because bioparts are typically characterised by relative metrics that vary across genetic and physiological contexts. To address this, we introduce a host-aware framework for quantitatively characterising bioparts in combinatorial libraries of plasmid-based constitutive expression constructs. The approach integrates a digital twin of Escherichia coli, conditioned on measured growth rate, with model-in-the-loop parameter identification to separate biopart-associated properties from host-dependent effects. Using structured combinatorial libraries, we identify mechanistically interpretable, transferable parameters for plasmid origins, promoters and ribosome binding sites. In particular, we define an intrinsic translation initiation capacity that captures the dominant RBS-associated contribution to translation while context-dependent expression emerges from host physiology and local sequence context. The resulting parameterisation accurately predicts protein synthesis across physiological conditions, supports incremental library expansion, and reveals localised failures of modularity, providing a scalable foundation for predictive host-aware design in synthetic biology.

Escherichia coli↗
PubMed1979

Isolation and characterization of the chicken ovomucoid gene.

The chicken ovomucoid gene has been isolated by screening a chicken DNA library with a plasmid containing ovomucoid mRNA sequences. Twelve recombinant phages carrying ovomucoid mRNA sequences were isolated. Two of them, extending farthest into the 5' and 3' direction respectively, were characterized by restriction mapping and Southern hybridization as well as by electron microscopic analysis of hybrids between the cloned DNA and ovomucoid mRNA. Seven intervening sequences interrupt the ovomucoid mRNA sequence in chromosomal DNA. From these data a minimal size of 5.6 kb can be estimated for the length of the ovomucoid gene.

Animals↗
PubMed2024

Identification of RBP binding sites using RNA deaminases.

RNA-binding proteins (RBPs) are critical regulators of gene expression and RNA processing. Identification of their binding sites has important implications for their physiological and disease-related functions. Crosslinking and immunoprecipitation, followed by sequencing (CLIP-seq) and its derivatives, are the most commonly used methods to identify RBP binding sites, but are laborious and require a large amount of starting material. Recent advancements harnessing RNA deaminases in fusion to any RBP of interest, allow for the profiling of RBP binding sites from low-input samples in simpler procedures. Among these efforts, we developed STAMP (Surveying Targets by APOBEC-Mediated Profiling), which efficiently detects RBP-RNA interactions. This chapter describes the detailed protocol for the STAMP method, including plasmid construction, delivery and sorting, library preparation and bioinformatic data analysis.

RNA-Binding Proteins↗
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↗
PubMed1979

Middle repetitive DNA: a fluid component of the Drosophila genome.

Most of the middle repetitive DNA of Drosophila melanogaster appears to be organized into families of 10-100 repeated elements that are found at scattered locations in the chromosome arms and occupy new chromosomal positions as populations of D. melanogaster diverge. These "nomadic" DNA segments can be identified by an analysis of cDm plasmids, hybrids of ColE1 and segments of randomly sheared D. melanogaster DNA. Eighty cDm plasmids were withdrawn, at random, from a library of approximately 17,000 cDm clones. Fifty-seven of these seem to contain either DNA that is not repeated in the D. melanogaster genome or DNA that has a low repetition frequency. The remaining 23 cDm plasmids contain repetitive sequences. Seventeen of these 23 plasmids contain repetitive sequences that are demonstrably scattered to many chromosomal sites that can be mapped in two D. melanogaster strains, g-1 and g-X11. The repeated elements hybridizing with each of the different Dm segments are at quite different chromosomal locations in these two strains. However, the size of each family of repeated sequences remains fairly constant in both strains. It is proposed that the number of elements in each family has been fixed by selection.

Animals↗
PubMed2026

Barcoded mutant library enables high-throughput functional genomics in a filamentous fungus.

Advances in sequencing technology enabling rapid and inexpensive whole-genome sequencing highlight how few genes are functionally characterized. This problem is particularly acute in filamentous fungi, where even in the best studied organisms upward of half of genes are poorly characterized or unannotated. High-throughput tools to identify gene function exist for single-celled organisms, like yeast and bacteria. However, filamentous fungi present challenges to high-throughput gene characterization, including low transformation efficiency and multinucleate cells. Filamentous fungi are critical components of nutrient cycling in ecosystems, form symbioses with plants that improve nutrient uptake, and are devastating human, plant, and animal pathogens causing millions of deaths and substantial crop loss each year. Thus, it is critical to overcome challenges to rapid gene characterization in filamentous fungi. We generated a library of hundreds of millions of uniquely barcoded plasmids containing a broad host-range drug resistance marker for ectopic insertion into filamentous fungal genomes by Agrobacterium tumefaciens. We then optimized A. tumefaciens mediated transformation of the biocontrol agent Trichoderma atroviride and made an insertional mutagenesis library containing 83,311 barcoded insertions, disrupting 5,331 of 11,863 predicted genes. This library enables high-throughput screens to rapidly connect genotype to phenotype. Quantifying relative barcode abundance in the pooled library before and after exposure to experimental conditions identified candidate genes and recovered known pathway components in amino acid biosynthetic, fructose utilization, and xylose utilization pathways. This resource establishes a scalable platform for high-throughput functional genomics in filamentous fungi, enabling investigations of fungal biology to improve medical outcomes, biotechnology, and sustainable agriculture.

Genomics↗
PubMed1979

Isolation of the lysozyme gene of chicken.

The lysozyme gene has been purified by molecular cloning from two chicken gene libraries. Several recombinant phages harbouring sequences homologous to a plasmid carrying a double stranded lysozyme cDNA have been isolated. One recombinant appears to carry an entire lysozyme gene. Electron microscopic studies show that the latter is split by at least three introns. The length of the gene is about 3.9 kb, 6 times longer than lysozyme mRNA.

Animals↗
PubMed1979

Structure of cloned ribosomal DNA cistrons from Bacillus thuringiensis.

A library of B. thuringiensis DNA has been prepared by using the plasmid pBR322 as a cloning vehicle and E. coli as a host cell. By screening this collection with specific probes, 17 clones were identified whose hybrid plasmids contain rRNA genes of B. thuringiensis. Several of these plasmids have been mapped with restriction endonucleases and by DNA-RNA hybridization. By using maps of overlapping fragments, we have been able to establish an overall map of the ribosomal gene cluster.

Bacillus thuringiensis↗
PubMed2026

High-throughput recovery of integron cassettes for gene discovery screens.

Integrons capture functional genes in mobile genetic elements called integron cassettes, which represent an untapped source of genes of biotechnological interest. Here we present two tools, cassette gatherer and cassette hunter, that enable high-throughput establishment of gene libraries either from genetically tractable strains or directly from DNA. We re-engineered a class 1 integron into counterselection markers on a plasmid or on the chromosome of a naturally competent Vibrio cholerae, which enabled capture of single cassettes in a sequence- and function-independent manner. When applied to Vibrio strains and genomic libraries, our tools recovered hundreds of single cassettes per assay with more than 99% specificity. We further subjected the library of cassettes generated by the hunter and gatherer tools to screens against phages ICP2 and T4, and identified nine phage-defence systems, including five previously undescribed. These tools enable rapid and large-scale recovery of integron cassettes that could be leveraged for functional gene discovery.

Journal Article↗
PubMed2005

SUMO modification of the Ets-related transcription factor ERM inhibits its transcriptional activity.

A variety of transcription factors are post-translationally modified by SUMO, a 97-residue ubiquitin-like protein bound covalently to the targeted lysine. Here we describe SUMO modification of the Ets family member ERM at positions 89, 263, 293, and 350. To investigate how SUMO modification affects the function of ERM, Ets-responsive intercellular adhesion molecule 1 (ICAM-1) and E74 reporter plasmids were employed to demonstrate that SUMO modification causes inhibition of ERM-dependent transcription without affecting the subcellular localization, stability, or DNA-binding capacity of the protein. When the adenoviral protein Gam1 or the SUMO protease SENP1 was used to inhibit the SUMO modification pathway, ERM-dependent transcription was de-repressed. These results demonstrate that ERM is subject to SUMO modification and that this post-translational modification causes inhibition of transcription-enhancing activity.

Adenoviridae↗
PubMed2025

Ultra-high field strength electroporation enables efficient DNA transformation and genome editing in nontuberculous mycobacteria.

Efficient DNA delivery is essential for genetic manipulation of mycobacteria and for dissecting their physiology, pathogenesis, and drug resistance. Although electroporation enables transformation efficiencies exceeding 10⁵ CFU per µg DNA in Mycobacterium smegmatis and Mycobacterium tuberculosis, it remains highly inefficient in many nontuberculous mycobacteria (NTM), including Mycobacterium abscessus. Here, we discovered that NTM such as M. abscessus exhibit exceptional tolerance to ultra-high electric field strengths and that hypertonic preconditioning partially protects cells from electroporation-induced damage. Using ultra-high electric field strength (3 kV/mm) electroporation, we achieved dramatic improvements in plasmid transformation efficiency-up to 106-fold in M. abscessus, 83-fold in Mycobacterium marinum, and 37-fold in Mycobacterium kansasii-compared to standard conditions (1.25  kV/mm). Transformation efficiency was further influenced by the choice of selectable marker. Ultra-high field strength electroporation also markedly enhanced allelic exchange in M. abscessus expressing Che9c RecET recombinases, increasing the recovery of gene deletion mutants by over 1,000-fold relative to conventional electroporation. In parallel, oligonucleotide-mediated recombineering for targeted point mutations produced nearly 10,000-fold more mutants under ultra-high field conditions. Together, these findings establish ultra-high field electroporation as a robust, broadly applicable platform for genetic engineering of NTMs. This method substantially enhances transformation efficiency and enables construction of advanced genetic tools-including expression libraries and CRISPRi knockdown libraries-in species that have historically resisted genetic manipulation.IMPORTANCEInfections caused by nontuberculous mycobacteria (NTM), including Mycobacterium abscessus, are increasing globally, yet genetic manipulation of these pathogens remains technically challenging due to inefficient DNA delivery and low gene editing success. The ultra-high electric field strength electroporation strategy described here overcomes these barriers, enabling dramatic improvements in both transformation and genome editing efficiency. This advance paves the way for high-throughput functional genomics in NTMs, including the construction of genome-wide knockout, CRISPRi knockdown, and expression libraries. Broad adoption of this approach will accelerate discovery of genetic determinants of virulence and drug resistance, facilitating the development of antimicrobials and vaccines.

Electroporation↗
PubMed2025

High throughput screening of eukaryotic release factor 1 variants to enhance noncanonical amino acid incorporation.

Noncanonical amino acids (ncAAs) enable diversification of protein functions, but the efficiency of genetic code expansion (GCE) in eukaryotes is hindered by competition between suppressor tRNAs and release factors. Prior work has identified eukaryotic release factor 1 (eRF1) mutants that improve ncAA incorporation, suggesting that screens for improved variants may lead to further enhancements. Here, we developed a high-throughput system to screen eRF1 mutants in Saccharomyces cerevisiae where eRF1 mutants are coexpressed on a plasmid alongside genomically encoded, wild-type eRF1. This strategy enabled recovery of live cells expressing eRF1 variants that enhance ncAA incorporation, even with mutants known to severely affect cell viability in the absence of WT eRF1 expression. We prepared and screened a million-member library of randomly mutated eRF1 variants for clones exhibiting improved ncAA integration phenotypes. Deep sequencing revealed a diverse set of enriched mutations across all three major domains of eRF1. Interestingly, several enriched mutations identified here are also found in naturally occurring eRF1 homologs from species that recode canonical stop codons. When eRF1 variants were combined with yeast knockout strains also known to enhance ncAA incorporation, this resulted in further improvements to efficiency, highlighting the complementarity of release factor engineering to other GCE enhancement strategies. This work demonstrates that high-throughput engineering of the eukaryotic translational apparatus is a powerful approach to identify previously unknown solutions for enhancing ncAA incorporation, with implications for elucidating and precisely manipulating the molecular functions of essential translational machinery.

Noncanonical amino acids↗
PubMed1979

The chicken conalbumin gene: studies of the organization of cloned DNAs.

The construction of a double-stranded conalbumin cDNA plasmid (1) has allowed us to investigate the structure of the conalbumin gene. Restriction enzyme mapping of chicken genomic DNA reveals that the conalbumin gene is split and is contained in three EcoRI fragments "a", "b", "b" and "c" which have sizes of 10.7, 4 and 2.5 kb, respectively. Analysis with specific probes shows that the orientation of these fragments with respect to transcription is 5'-"b", "c" and "a"-3. The fragments Eco "b", Eco "c" and part of Eco "a" have been isolated by molecular cloning from three different "libraries". Electron microscopic studies of hybrids between cloned DNA's and conalbumin mRNA show that one of the isolated clones, lambda C4-conl, contains the coding sequences for the first 940 nucleotides of the mRNA (out of 2400). This region is highly split, since it contains seven short exonic sequences separated by six intervening sequences. The DNA region coding for these 940 nucleotides is 5 times longer than the mRNA coding sequences, a ratio very similar to that found for other chicken genes.

Animals↗
PubMed2026

A combinatorial construct library enables an expanded expression range of secreted therapeutic proteins by probiotic yeast.

Orally administered engineered probiotics, including Saccharomyces cerevisiae var. boulardii (Sb), are of emerging interest as protein therapeutic delivery platforms to treat gastrointestinal diseases. Tools to readily optimize protein output are required to optimize the therapeutic index of Sb-produced therapies. In this study, a 125-plex Sb secretion construct library was developed consisting of all possible combinations of five promoters, five secretion signals, and five terminators, which enabled a greater than 1800-fold range in Sb expression of a Gaussia luciferase (GLuc) reporter. Secretion signal and promoter identities had significant effects on secretion output. This library further enabled a 28-fold improvement of binding activity of Sb-secreted haPD-1, an established anti-tumor immunotherapeutic, and improved haPD-1 detection in mouse stool samples following oral gavage of Sb_haPD-1. Sb secretion trends of both GLuc and haPD-1 in vitro mirrored payload expression in vivo. This protein secretion library toolkit will serve as a valuable resource to rapidly optimize protein therapeutic output from engineered Sb.

Saccharomyces boulardii↗