BMC Biotechnology

Plant viruses are recognized as rapid and effective vectors to deliver CRISPR-Cas reaction components into plants, a strategy termed virus-induced gene editing (VIGE). However, VIGE is limited by the host range of the viral vectors. Development of new viral vectors to target a broad range of plant species will potentially enable the delivery of the editing components to new cultivars. Potyviruses (genus Potyvirus) comprises the largest group of plant RNA viruses. The main limitation of potyviral vectors to express a non-coding RNA consists of potential insertion of stop codons that interrupt the large open reading frame that encompass most potyviral genome. This is the case with the Streptococcus pyogenes Cas9 sgRNA scaffold, which contains stop codons in all three possible frames. In this work, we first built on a visual reporter system targeting the two homeologs of Nicotiana benthamiana Magnesium chelatase subunit I (CHLI). Second, we developed a tobacco etch virus (genus Potyvirus)-derived vector for VIGE by engineering a modified Cas9 scaffold, free of stop codons, to maintain the potyviral polyprotein reading frame while ensuring effective editing. This vector self-replicates and moves systemically, delivering sgRNAs efficiently throughout the plant. This allowed to obtain plants exhibiting a white phenotype with their four alleles edited through in vitro regeneration from infected leaves, and also to produce edited progeny. We further demonstrated the vector utility in tomato. Given the conserved biological properties within the genus Potyvirus, these findings must be broadly applicable to other potyviruses, expanding the reach of the VIGE technology.

It is commonplace in East Africa for 100% of cassava fields to be infected with Cassava mosaic disease (CMD) and/or Cassava brown streak disease (CBSD), resulting in annual losses of more than US$1.25 billion and reduced food and economic security for farming households. The vector of both diseases is the African cassava species of the whitefly Bemisia tabaci. Since the late 1990s, there has been an unprecedented increase in whitefly populations, to the extent that they are referred to as "super-abundant". Research efforts since the late 1990s has focused mainly on developing plant resistance to the viral pathogens and paid scant attention to understanding the root causes of disease epidemics or the control of whitefly infestation. Here, we aimed at developing long-term whitefly-control solutions using an in-planta RNA interference (RNAi) approach. First, transcriptome analysis identified candidate genes that play key roles in whitefly biology: osmoregulation, sugar metabolism and transport, symbiosis with endosymbiotic bacteria and detoxification of phytotoxins. Then, fifteen RNAi inverted repeat constructs were produced, designed to target the candidate genes and 140 independent transgenic lines were generated in cassava variety NASE 13. Whole plant bioassays showed insecticidal activity of transgenic plants, reaching 58% lethality for adults within 7 days and 75-90% lethality of nymphs after 25 days, compared to control plants. Target genes were confirmed to be downregulated by up to 2.5-fold in adult whiteflies and nymphs. We used population dynamics modelling to predict the potential of the RNAi technology to control whiteflies under field conditions in East Africa.

RNA interference (RNAi) shows great potential to protect crops against fungal diseases, yet reported protection efficiencies vary greatly, and our understanding of the factors responsible for this variance remains limited. In this meta-analysis, we evaluated 89 studies that compare the efficiency of host-induced gene silencing (HIGS) and spray-induced gene silencing (SIGS) in controlling fungal diseases, focusing on biotrophic, hemibiotrophic, and necrotrophic fungi, the use of formulations, and the dsRNA design as explanatory factors for differences between reported efficiency values. Our results indicate that SIGS is slightly more effective, particularly in biotrophs. Surprisingly, SIGS studies using formulations did not outperform those applying naked dsRNA. We also assessed parameters of RNA design. Differences in dsRNA length and the number of constructs, and number of targets showed no consistent significant effect on resistance in either HIGS or SIGS. Interestingly, however, HIGS studies reported significantly higher efficiency when targeting genes closer to the 3 end and SIGS when targeting genes closer to the 5 end. We discuss potential reasons for the reported patterns, such as variability in dsRNA uptake mechanisms, intercellular trafficking and Dicer processing, and conclude that more research is needed to understand the biological mechanisms determining RNAi efficiency for fungal control.

Cultivated meat has undeniable potential to address some of the current detrimental impacts of animal farming, while addressing food security worldwide. However, one of the main challenges in cultivated meat production is manufacturing cost. The main contributor to cost is the culture media which comprises expensive components such as growth factors and animal-derived proteins. This study investigated alternative, food grade, high protein extracts as serum replacements in serum-free media formulations. The extracts were chosen to represent various sustainable sources of proteins: marine (spirulina e.g. cyanobacterium), plant (faba bean) and insect (mealworm flour). Different processing methods and different solvents were investigated for production of cell culture-compatible extracts which were then tested with mouse myoblasts (C2C12) and primary porcine myosatellites (pMyoSCs). A serum-free medium formulation containing 2.6% v/v spirulina extract was found to support long term growth of C2C12 cells for [~]10 population doublings compared to only [~]2 in the control. The processing steps were optimized, showing that a glycerine solution was best for free amino acid and protein yield (4950 {micro}M total free amino acids, 11.45 mg/mL protein concentration). This solution had a positive effect on C2C12 cells, increasing their growth by up to 20% when added to the B8 medium. However, this benefit did not translate to pMyoSCs, which showed no significant growth increases in short-term screening. This work demonstrates a method for converting food grade protein powders into effective culture media supplements and highlights the potential of spirulina-based extracts for the use in cultivated meat. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=104 SRC="FIGDIR/small/702276v1_ufig1.gif" ALT="Figure 1"> View larger version (24K): org.highwire.dtl.DTLVardef@206658org.highwire.dtl.DTLVardef@11f28ceorg.highwire.dtl.DTLVardef@b00fd6org.highwire.dtl.DTLVardef@dfaaf4_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphical abstractC_FLOATNO Created in BioRender. Gordon-Petrovskii, W. (2025) https://BioRender.com/by7khs1 C_FIG

Gene therapy offers the potential for long-term treatment or cures for a range of chronic diseases. However, permanent gene therapy expression may not be desirable. Efforts have been made to create systems which can be switched on/off by stimuli including light, designer drugs, or cellular contexts such as increased electrical activity. Here, we designed a novel plasmid system in which ion channel expression, and therefore function, is regulated by microRNA (miR) - an endogenous class of short noncoding RNAs which negatively regulate gene expression via binding the 3 untranslated region of target transcripts. We modified an existing voltage-gated potassium channel gene therapy with a binding cassette for miR-193a-3p, and transfected this miR-193-OFF system in neuro2A cells. Co-transfection with an inhibitor or mimic of miR-193a-3p respectively enhanced or repressed expression of our transgene, assessed using a GFP marker. Using whole-cell voltage clamp, we observed enhanced voltage-gated potassium currents in cells co-transfected with a miR-193a-3p inhibitor, compared with a non-targeting control oligonucleotide. Together, this demonstrates the concept of a novel miR-mediated molecular switch which can bias therapeutic ion channel expression based on a specific miR signal. As miRs are a ubiquitous molecular mechanism, our approach could be applied to a wide range of cellular and disease contexts, potentially expanding gene therapy to new patient populations.

BACKGROUNDInsect pests present a global threat to crops, with plant resistance representing a key breeding goal. The cabbage stem flea beetle (Psylliodes chrysocephala; CSFB) is the most damaging pest of oilseed rape (Brassica napus; OSR) within Europe; however, CSFB resistance is yet to be found within B. napus. To address this, we examine CSFB larval development over time, explore antibiosis across a diverse Brassica panel, and test whether larvae can develop within model Brassica relatives (Brassica rapa and Arabidopsis thaliana). RESULTSCSFB larvae completed development from four weeks post-infestation, undergoing a 20-fold size increase, with larval recovery after two weeks allowing semi-high throughput resistance phenotyping. Applying this method to 98 Brassica genotypes (97 B. napus and a single Sinapis alba), we found weak evidence for genotype effects on larval survival, however phenotype validation with resistant and susceptible B. napus genotypes showed no differences in larval survival or adult emergence. Larval antibiosis was consistently observed in S. alba. Finally, we showed that B. rapa and A. thaliana represent suitable hosts for CSFB, with larvae increasing 8-10x in size after two weeks. CONCLUSIONCSFB larval antibiosis appears absent within B. napus, possibly due to bottlenecks experienced during domestication. However, larval antibiosis is present within S. alba, and future work should study the basis of this resistance. Further, CSFB larval screening in Brassica relatives presents an opportunity to explore CSFB resistance genetics, informing breeding progress for insect resistance in B. napus.

Collecting cells from zebrafish embryos for genotyping is critical to rapid research with these model organisms. The standard collection process is manual, labor-intensive, time-consuming, and requires a skilled person to perform it. To overcome this challenge, researchers are exploring the development of automated genotyping tools for live animals, which would significantly enhance the efficiency and accuracy of genetic screening in zebrafish and other species. The focus of this research was to optimize the Zebrafish Embryo Genotyper (ZEG), an automated system used for the rapid extraction of cellular material from zebrafish embryos. This system rapidly vibrates a roughened chip containing a zebrafish embryo to collect genetic material safely and efficiently. The aim was to improve the efficiency of DNA collection from the chips used with the ZEG by identifying the key factors that contribute to the process. First, the chips were modified to resolve issues associated with loss of sample volume from the chip wells due to evaporation during processing. Second, we experimented with three critical parameters - sample volume in the wells, the vibrational frequency of the system, and the operation time - on the quantity of DNA collected. The performance was evaluated by measuring embryo survival and quantifying the DNA collected. The sensitivity (previously 90%) of the DNA collection and embryo survival (previously 95%) of the were both found to be greater than 95% after optimization. The optimized design parameters (15 {micro}L solution volume, 2.4 V, and a 5-minute run with 5 s alternating on/off) provided a >50% increase in DNA collection compared to the previous designs and parameters. The proposed chip design and operation do not appear to cause any apparent adverse effects on the development or survival of the embryos.

European ash dieback caused by the invasive ascomycete species Hymenoscyphus fraxineus poses the most prominent danger to common ash trees (Fraxinus excelsior) in Europe. The disease is widely distributed in Europe and currently no efficient management strategy is available. Host-induced gene silencing and exogenous dsRNA applications have shown great potential for controlling fungal diseases in crop plants. In this study, we reported in silico evidence for the presence of a functional RNA interference pathway in Hymenoscyphus fraxineus. Moreover, we showed that the transgenic expression of a double stranded RNA (dsRNA) leads to inhibition of translation of its target polyketide synthase-like gene, a fungal endogene. We explored whether the dsRNA could be introduced exogenously and demonstrated that H. fraxineus can take up externally applied dsRNA molecules. This study highlights the RNA interference mechanism in H. fraxineus and suggests exoRNA applications as a promising approach to control European ash dieback.

We have developed a vector platform for delivery of foreign peptides by genetic modification of the temperate lambda ({lambda}) bacteriophage. This delivery platform is capable of displaying peptides or proteins on either terminus of the structural {lambda} head protein D, present in [~]420 copies per phage particle, and {lambda} side tail fiber (Stf), present at 12 copies per phage particle. Proteins and peptides can be easily fused for display through the low-cost and high-efficiency methods of recombineering and {lambda} prophage induction for recombinant phage preparation described here. To improve this vector technology for use in antigen selection and immunotherapy, we introduced several mutations in the bacterial host and resident prophage {lambda} that improve engineering, induction, phage stability, yield, fusion protein accommodation capacity, and longevity in animal systems. We tested the ability of this {lambda} display system to identify useful antigens and generate antibodies in a mouse model. We report its success as a new technology for both applications: the selection and delivery of therapeutic peptides and proteins.

T/A cloning is a popular method for generating recombinant DNA plasmids. This method relies on single A:T nucleotide base pairs between PCR product ends and vector. Theoretically, the directionality of insert ligation with relation to the vector is random. However, we have continuously observed directionality bias using the pGEM-T Vector System for T/A cloning in a Course-based Undergraduate Research Experience (CURE). Cloning of over 400 inserts has shown directional bias higher than 74% (p-value < 0.0005) "sense" to the T7 promoter of the vector. Awareness of biased insertion in our applications reduces time and cost in cloning and downstream analyses.

Efficient production of human proteins for the development of tool compounds and biologics depends on a detailed understanding of the protein expression machinery in mammalian cells. Codon optimization is widely believed to enhance protein yield, yet its impact in homologous mammalian systems remains poorly defined. Here, we systematically compare five codon usage strategies reflecting common assumptions about rare codons, RNA stability, and synthesis efficiency. We developed pTipi, an efficient open-source mammalian expression vector, and evaluated its performance in antibody production. We generated plasmids for common epitope tag antibodies such as V5, anti-biotin and anti-His for distribution by Addgene. To compare codon usage schemes, we performed a bake-off of 18 human and murine Wnt pathway glycoproteins in mammalian cells. Small-scale expression screens revealed that codon optimization did not provide a general advantage over native coding sequences, while strategies prioritizing RNA stability consistently reduced expression. Interestingly, a skewed codon scheme using the most abundant codons produced yields comparable to native sequences and occasionally enhanced protein output. To enable flexible evaluation of codon strategies, we implemented a Golden Gate-compatible pTipi platform for efficient synthetic gene incorporation. We conclude that native codons are sufficient for robust homologous mammalian expression of glycoproteins, while selective codon skewing can be beneficial for some targets.

Peanut (Arachis hypogaea L.), a vital oilseed and food legume, is cultivated across the globe. Genetic improvement via conventional breeding faces limitations from narrow diversity and reproductive barriers, underscoring the need for tissue culture-based regeneration and transformation platforms. This study optimizes an efficient, reproducible in vitro regeneration protocol using cotyledonary node explants from three Indian elite cultivars: GG-20, GJG-9, and TAG-37A. Explants from aseptically germinated seedlings were cultured on Murashige and Skoog (MS) medium with varying cytokinins (e.g., BAP 0-4 mg/L) and auxins (e.g., NAA 0.1-0.9mg/L), yielding direct multiple shoot induction without callus, minimizing somaclonal variation. Optimal shoot proliferation occurred on full-strength MS + 2 mg/L BAP for GG-20/GJG-9 (88.9% efficiency) and 4 mg/L BAP for TAG-37A ([~]89-100% efficiency); rooting peaked on half-MS + NAA (up to 88.9% in GG-20). Regenerated plants acclimatized successfully in greenhouse conditions. Additionally, a robust in planta Agrobacterium tumefaciens (EHA105, pGFPGUSPlus) transformation via plumular meristem pricking in GG-20 achieved 7.69% efficiency. Transgene integration was confirmed by GUS assay and PCR (GUS/hptII), with [~]64% soil establishment.

Transgenic mouse lines are essential to uncover organ or system level genotype-phenotype relationships. The generation of such lines via transgene addition may lead to the insertion into unknown genomic loci potentially leading not only to the disruption of native genes but also attenuation of transgene expression. Additionally, this often results in the inability to determine transgene zygosity which in turn complicates breeding and interpretation of experimental results. In this study we present two whole genome sequencing based pipelines that allow the identification and genotyping of even complex multi transgenic inserts. As they use widely available reagents and bioinformatic tools, they can easily be applied to develop genotyping strategies in potentially any species.

The composition of culture medium is a major, yet frequently undercontrolled, determinant of hepatic cell state in vitro. For decades, fetal bovine serum (FBS) has been routinely incorporated into liver cell culture. Its undefined and lot-to-lot variable composition can, however, confound cell identity and experimental reproducibility. Serum-free, chemically defined media (CDM) represent an alternative approach that can improve standardization, but the consequences of transitioning from FBS-supplemented media (FBS-SM) to CDM remain insufficiently characterized in hepatic models, particularly with respect to metabolic and detoxification programs that govern xenobiotic handling and hepatotoxicity readouts. Here, we systematically assessed how replacing FBS-SM with CDM remodels transcriptomic profiles in two widely used human hepatic cell lines (HepaRG and HuH7 cells) and compared the results to that obtained from primary human hepatocytes (PHH). Global transcriptomic analyses indicated that cell type was the primary driver of variance, whereas medium induced a model-dependent secondary effect. Functional interpretation showed preferential enhancement of xenobiotic metabolism and transport-associated programs in HepaRG cells, while HuH7 cells response was dominated by lipid/sterol homeostasis and stress-linked processes. Benchmarking against PHH based on hepatic identity and detoxification gene panels further supported improved PHH alignment for HepaRG cells under CDM compared to cultures with FBS-SM, with limited improvement for HuH7 cells. Collectively, these findings address a key knowledge gap by defining how FBS-SM and CDM impact the transcriptomic profiles of HepaRG and HuH7 cells.

BackgroundThe cotton bollworm Helicoverpa armigera is a major global pest controlled by genetically engineered crops expressing Bacillus thuringiensis (Bt) toxins, including Vip3Aa. While Vip3Aa is widely deployed, the genetic basis of resistance remains poorly understood. Previous work identified disruption of a thyroglobulin-like gene (HaVipR1) as one mechanism of resistance, suggesting additional loci may be involved. ResultsUsing linkage analysis, transcriptomics, long-read sequencing, and CRISPR-Cas9 gene editing, we identify a second thyroglobulin-like gene, HaVipR2, as a novel mediator of Vip3Aa resistance. Resistance in a field-derived H. armigera line was shown to be monogenic, recessive, and autosomal, mapping to chromosome 29. Long-read sequencing revealed a [~]16 kb transposable element insertion disrupting HaVipR2, which was undetectable using standard short-read approaches. CRISPR-Cas9 knockout of HaVipR2 conferred >900-fold resistance, confirming its causal role. Comparative analyses show that HaVipR1 and HaVipR2 share conserved domain architecture, indicating that thyroglobulin-domain proteins represent a recurrent target of resistance evolution. ConclusionsOur findings establish thyroglobulin-domain proteins as a new class of Bt resistance genes in Lepidoptera and demonstrate that transposable element insertions can drive adaptive resistance while evading detection by conventional methods. These results highlight the importance of long-read sequencing and accurate genome annotation for resistance monitoring and provide new insights into the molecular basis and evolution of Vip3Aa resistance.

Virus induced gene silencing (VIGS) is a method that exploits plant antiviral defence mechanisms to downregulate endogenous genes. The technique is versatile, rapid, and widely used for functional genomics studies. Here we report a method for VIGS in the medicinal plant, Calendula officinalis (pot marigold). This species produces anti-inflammatory triterpenoids and has also been bred and cultivated as an ornamental plant. We describe a method for the injection of Agrobacterium tumefaciens cultures into leaf midribs and compare visual marker genes for tracking VIGS utilising constructs that simultaneously target visual marker and target genes. We use these tools to demonstrate that silencing a gene encoding cycloartenol synthase results in changes to leaf phytosterols. This method could be used to further investigate the genetic basis of specialised metabolism in this species and could be adapted to other members of the Asteraceae family, many of which are of economical and chemical value.

Choanoflagellate genetics has undergone rapid and impactful developments in the last decade. Currently, the primary method for genetic modification of choanoflagellates relies on proprietary nucleofection reagents to deliver transgenes for ectopic expression or CRISPR-Cas9 ribonucleoprotein complexes for targeted genome editing. The acquisition of proprietary buffers required for nucleofection can hamper advances in choanoflagellate research due to costs, shipping limitations, and restrictions that prevent buffer components from being optimized for understudied organisms. Therefore, we test whether a low-cost in-house electroporation buffer developed for other systems can replace the proprietary buffer currently used for choanoflagellate transfection. Here, we present an in-house buffer with transfection efficiency comparable to that of the previously established proprietary buffer. This work increases the accessibility of choanoflagellate genetics and can broaden research participation in investigating animal origins.

Precision-cut lung slices (PCLS) have emerged as a powerful tool for studying the biology of viable human lung tissue. However, the presence of agarose impurities compromises RNA yield and integrity during the extraction process. We tested whether using an alternative Plant kit RNA extraction method to wash agarose impurities or pre-dissolving agarose from PCLS implementing a dissolving buffer for routine RNA isolation in gel-electrophoresis would improve RNA quantity, quality, and integrity. Our results show that RNA quantity and integrity are highly compromised when using a conventional method of RNA extraction. The plant kit and dissolution of agarose increased the RNA quantity to 0.42{+/-}0.11 and 0.65{+/-}0.17 {micro}g/PCLS (measured by the Qubit) and integrity number to 6.60{+/-}0.59 and 9.13{+/-}0.39 (measured by the Bioanalyzer), respectively. The presence of impurities in conventional and Plant kit extractions misled to an overestimation of the RNA quantity and quality using the NanoDrop. The Plant kit and agarose dissolution showed a significant transcript integrity increase in GUSB (p<0.0001) and COL1A1 (p<0.05) expression, validating these methods over conventional extraction. We encourage laboratories applying PCLS experimentation to implement alternative methods to remove agarose impurities during RNA extraction, as well as to rely on sensitive quantitative techniques, such as the Qubit and Bioanalyzer, for RNA quantification and integrity measurements.

Particle bombardment systems are widely used for plant transformation, but commercial devices are expensive and rely on high-pressure helium gas. This study aimed to develop a cost-effective and helium gas-free alternative using an air duster gun connected to a commercial compressor. A nozzle (for DNA with transgenes), gold particles (as DNA carriers), nozzle-to-sample distance, and a method for coating gold particles with DNA were optimized to yield better transformation efficiency in targeting onion epidermal cells and rice calli. From the rice calli transformed with the newly developed system (a tool to shoot genes with massive air from a compressor: TSGMAC), stable transgenic plants could be obtained. TSGMAC offers a low-cost and helium gas-free solution for plant transformation and genome editing and can enhance accessibility to particle bombardment-based techniques.

Mammalian cell lines are the preferred hosts for producing commercially relevant therapeutic proteins such as antibodies, multispecifics, and cytokine fusion proteins. Even though significant investment is made to optimize upstream and downstream processes, the optimal gene design parameters for heterologous recombinant protein expression remain poorly understood. We describe here a generic approach to gene optimization in which design features are systematically sampled and modulated iteratively using machine learning (ML). Synthetic genes encoding the Dasher fluorescent protein, differing only in synonymous codons, were used to interrogate the gene-sequence preferences of transient antibody-expressing HEK293 cells. Synonymous codon variations influenced expression by more than two orders of magnitude. This variation in protein yield was used to build ML models relating gene design features, which were then employed to design further-improved genes. The ML models were shown to be expression system-specific. Messenger RNA levels and ribosome occupancy were highly correlated with protein levels, suggesting that mRNA lifetime has a causal relationship with coding bias. Our results illustrate a novel, generally applicable method to improve gene expression via synonymous re-coding for any protein target or host cell.