BMC Biotechnology

The ability to control gene expression is pivotal in genetic engineering and synthetic biology. However, in most non-model and pest insect species, empirical evidence for predictable modulation of gene expression levels is lacking. This knowledge gap is critical for genetic control systems, particularly in mosquitoes, where transgenic methods offer novel routes for pest control. Commonly, the choice of RNA polymerase II promoter (Pol II) is the primary method for controlling gene expression, but the options are limited. To address this, we developed a systematic approach to characterize modifications in translation initiation sequences (TIS) and 3 untranslated regions (UTR) of transgenes, enabling the creation of a toolbox for gene expression modulation in mosquitoes and potentially other insects. The approach demonstrated highly predictable gene expression changes across various cell lines and promoter sequences, representing a significant advancement in mosquito synthetic biology gene expression. tools. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=100 SRC="FIGDIR/small/555539v1_ufig1.gif" ALT="Figure 1"> View larger version (31K): org.highwire.dtl.DTLVardef@1db954forg.highwire.dtl.DTLVardef@1279543org.highwire.dtl.DTLVardef@1b86fa2org.highwire.dtl.DTLVardef@1a9176a_HPS_FORMAT_FIGEXP M_FIG C_FIG

We report the identification and functional validation of a 7SK RNA polymerase III promoter in the Mediterranean fruit fly, Ceratitis capitata. CRISPR/Cas9-based genetic control strategies for this global agricultural pest, including gene drives and precision guided sterile insect approaches, require efficient guide RNA expression, yet only a single U6 Pol III promoter had previously been validated for this purpose in C. capitata, and no 7SK promoter had been characterised in any Tephritid species. Using comparative genomics with Drosophila orthologues, we identified a previously unannotated 7SK gene in the C. capitata genome, confirmed its transcriptional activity by RT-PCR, and demonstrated that the cloned promoter drives functional guide RNA expression in CRISPR/Cas9-mediated knockouts of the white gene. Comparative analysis identified putative 7SK orthologues across the Tephritid fruit flies. The availability of this additional new Pol III promoter will enable multiplexed guide RNA strategies using distinct promoters, supporting more robust genetic control designs. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=89 SRC="FIGDIR/small/719894v1_ufig1.gif" ALT="Figure 1"> View larger version (30K): org.highwire.dtl.DTLVardef@156c203org.highwire.dtl.DTLVardef@db5eedorg.highwire.dtl.DTLVardef@353adforg.highwire.dtl.DTLVardef@ac079a_HPS_FORMAT_FIGEXP M_FIG C_FIG

BackgroundThe black soldier fly (BSF), Hermetia illucens is a widely used, and mass-produced insect that fulfils an important role in both the management of organic waste and as a component of animal feed formulations. They also have significant potential as a platform for converting organic waste into high-value proteins, and lipids for the production of biofuels. Applying synthetic biology to BSF provides even more potential for improvement through the generation of transgenic BSF to enhance animal feed, produce and fine tune high-value industrial biomolecules, and to expand their waste conversion capabilities. ResultsTo enable the rapid generation and screening of transgenic BSF, we utilised microinjections of piggyBac mRNA with donor plasmids. We have found preliminary screening of G0 BSF to identify mosaics for outcrossing can be completed less than 2 weeks after microinjection. Stable transgenic lines were reliably generated with effective transformation rates of 30-33%, and transmission of the transgene could be confirmed 3 days after outcrossing the G0 adults. We also present a protocol for identifying the location of integrated transgenes. ConclusionsThe methods presented here expedite the screening process for BSF transgenesis and further expand the toolkit for BSF synthetic biology.

Culex quinquefasciatus Say is a mosquito which acts as a vector for numerous diseases including West Nile Virus, lymphatic filariasis and avian malaria, over a broad geographical range. As the effectiveness of insecticidal mosquito control methods declines, the need has grown to develop genetic control methods to curb the spread of disease. The piggyBac transposon system - the most widely used genetic transformation tool in insects, including mosquitoes - generates quasi-random insertions of donor DNA into the host genome. However, despite the broad reported species range of piggyBac, previous attempts to use this tool to transform Culex quinquefasciatus mosquitoes have failed. Here we report the first successful transformation of Culex quinquefasciatus with the piggyBac transposon system. Using commercially synthesised piggyBac mRNA as a transposase source, we were able to generate three independent insertions of a ZsGreen fluorescent marker gene, with transformation efficiencies of up to 5%. Through this work, we have expanded the genetic toolkit available for the genetic manipulation of Culex mosquitoes and thus removed a barrier to developing novel genetic control methods in this important disease vector.

Mouse genome modification requires costly equipment and highly skilled personnel to manipulate zygotes. A number of zygote electroporation techniques were reported to be highly efficient in gene delivery. One of these methods called i-GONAD (improved Genome-editing via Oviductal Nucleic Acids Delivery) describes electroporation-based gene transfer to zygotes in utero. Here we adopted this technology for mouse genome-editing from scratch minimizing the cost of equipment, operator skill and animal use. We chose the CRISPR/Cas9 system as a genome editing tool and i-GONAD as a gene delivery method to produce IL10 gene knockout in C57BL/6 mice. Four animals out of 13 delivered pups (30.8%) were genetically compromised at IL10 gene locus suggesting the feasibility of the approach. This report provides one of the possible technical settings for those who aim at establishing in-house mouse transgenesis pipeline at minimal cost from scratch. For citationPopova J.V., Bets V.D., Omelina E.S., Boldyreva L.V., Kozhevnikova E.N. Mouse Genome Editing from Scratch. Journal of Experimental Biology. Year; issue (number): page. DOI

Genetic manipulation of Aedes aegypti is key to developing a deeper understanding of this insects biology, vector-virus interactions and makes future genetic control strategies possible. Despite some advances, this process remains laborious and requires highly skilled researchers and specialist equipment. Here we present two improved methods for genetic manipulation in this species. Use of transgenic lines which express Cre recombinase allowed, by simple crossing schemes, germline or somatic recombination of transgenes, which could be utilized for numerous genetic manipulations. PhiC31 integrase based methods for site-specific integration of genetic elements was also improved, by developing a plasmid which expresses PhiC31 when injected into early embryos, eliminating the need to use costly and unstable mRNA as is the current standard.

BackgroundA basic requirement for synthetic biology is the availability of efficient DNA assembly methods. Numerous methods have been previously reported to accomplish this task. One such method has been reported, which allows parallel assembly of multiple DNA fragments in a one-tube reaction, called Golden Gate Assembly. This study aims to further simplify that method and make it more suitable for small labs and students. MethodsPrior to amplification of the parental plasmids used in building the modules were domesticated using a variation of SDM (Site Directed Mutagenesis) called SPRIP. After careful design and amplification of the desired modules, using a high-fidelity polymerase, amplified PCR fragments that enter the one-step-one-pot reaction were stored in Zymo DNA/RNA Shield at -20 degrees C and thawed whenever needed to be used as fragments or modules in the assembly. The fragments were designed to posses unique overhangs using NEB Golden Gate assembly tool and Snapgene, amplification of modules was performed using a Q5 high fidelity polymerase from preexisting plasmids or gene fragments, clean-up of the PCR products (fragments) was performed in one tube per assembly using Zymo DNA Clean and Concentrator-5, assembled using BsaI and T4 ligase, DpnI digestion performed for eliminating the background plasmids that remain after the PCR reaction and the resulting assembled product was transformed into competent E.coli cells. Transformants were screened using diagnostic digest, transfected into HEK293T cells and the fluorescence was evaluated using fluorescent microscopy and flow cytometry. ResultsHerein presented is a simple and inexpensive alternate protocol to build modular plasmids using the Golden Gate Assembly method. A total of p37 S/MARs mammalian expression vectors were designed and constructed using 6 modules previously amplified by PCR and stored in the appropriate buffer to eliminate exo- and endonuclease activity and to protect the DNA from freeze thaw cycles. The existing modules were interchangeable and new modules were easily amplified and stored for use when needed. The mammalian expression vectors constructed showed the desired restriction pattern and GFP expression in bacteria and in mammalian cells. A comparison of 7 pNoname variants was conducted using flow cytometry. Interestingly, no pNoname variant harbouring the SV40 promoter showed expression in tested HEK293T cells. It appears that using the Ef1a promoter in combination with the BGH polyA signal provides the best expression in S/MARS vectors harboring the DTS40 region, as measured by flow cytometry. ConclusionsProvided the design steps are respected and the fragments are stored and labeled appropriately, multiple plasmid variants and combinations of the pre-designed modules can be assembled in one day, easier and using less resources than the established protocols, with good efficiency. The simplicity of the design and the affordability of the method could make modular cloning of plasmid constructs more accessible to small labs and students.

ABSTRACTAbility to functionally screen gene regulatory sequences, such as promoters and enhancers, in high throughput manner is an important prerequisite for many basic and translational research programs. One of the methods that allow such screening is STARR-seq, or self-transcribing active regulatory region sequencing. It allows to quickly screen millions of candidate sequences in the cell type of interest. However, it does rely on transfection as a delivery method which can be a limiting-step for some hard-to-transfect cells such as senescent cells. Here we show that integration-deficient and integration-competent promoterless lentiviral particles can be used to deliver STARR-seq constructs into cells. These constructs reported CMV enhancer activity both at protein and mRNA level. While further validations are necessary, ability to deliver STARR-seq libraries using lentiviral particles will significantly improve the versatility and usability of such a method.Competing Interest StatementThe authors have declared no competing interest.View Full Text

Culex quinquefasciatus Say is a brown, medium sized mosquito distributed widely in both tropical and subtropical regions of the world. It is a night-active, opportunistic blood-feeder and is responsible for vectoring many animal and human diseases, including West Nile Virus and avian malaria. Current vector control methods (e.g. physical / chemical) are increasingly ineffective; use of insecticides also imposes some hazards to both human and ecosystem health. Recent advances in genome editing have allowed the development of genetic methods of insect control, which is species-specific and, theoretically, highly effective. CRISPR/Cas9 is a bacteria-derived programmable gene editing tool that has been shown to be functional in a range of species. We demonstrate here, the first successful germline gene knock-in by homology dependent repair in C. quinquefasciatus. Using CRISPR/Cas9, we integrated exogenous sequence comprising a sgRNA expression cassette and marker gene encoding a fluorescent protein fluorophore (Hr5/IE1-DsRed, Cq7SK-sgRNA) into the kynurenine 3-monooxygenase (kmo) gene. We achieved a minimum transformation rate of 2.8% similar to rates achieved in other mosquito species. Precise knock-in at the intended locus was confirmed by sequencing. Insertion homozygotes displayed a white eye phenotype in early-mid stage larvae and a recessive lethal phenotype by pupation. This work shows an alternative and efficient method for genetic engineering of C. quinquefasciatus, providing a new tool for researchers interested in developing genetic control tools for this vector.

The larvae of the greater waxmoth, Galleria mellonella, are gaining prominence as a versatile non-mammalian in vivo model to study host-pathogen interactions. Their ability to be maintained at 37{degrees}C, coupled with a broad susceptibility to human pathogens and a distinct melanisation response that serves as a visual indicator for larval health, positions Galleria as a powerful resource for infection research. Despite these advantages, the lack of genetic tools, such as those available for zebrafish and fruit flies, has hindered development of the full potential of Galleria as a model organism. In this study, we describe a robust methodology for generating transgenic Galleria using the PiggyBac transposon system and for precise gene knockouts via CRISPR/Cas9 technology. These advances significantly enhance the utility of Galleria in molecular research, opening the way to its widespread use as an inexpensive and ethically compatible animal model for infection biology and beyond.

The ability to influence the sex ratio of mammalian offspring has applications in agriculture and animal welfare. Here we describe a genetic system in mice that produces predominantly female progeny, reaching approximately ninety percent, without affecting litter size or introducing genetic modifications into the offspring. The system consists of a doxycycline-regulated cassette inserted into the Y chromosome, encoding dCas9 and RNA guides intended to impair the function of Y bearing sperm. While the cassette was originally intended to repress a spermatid maturation gene, our analyses did not detect meaningful changes in gene expression or sperm function that would account for the phenotype. Nevertheless, male mice carrying the cassette consistently produced female biased litters, and the effect was reversed by doxycycline, confirming that it depends on cassette expression. Similar female bias was observed following in vitro fertilization using sperm from transgenic males. These findings demonstrate a reproducible, genetically confined, and conditionally regulated system for sex ratio bias in mice. The strategy may prove adaptable across species, even without a complete mechanistic understanding. Graphical AbstractA single line genetic system producing female biased, genetically unmodified progeny in mice by conditionally impairing Y bearing sperm. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=130 SRC="FIGDIR/small/568055v4_ufig1.gif" ALT="Figure 1"> View larger version (22K): org.highwire.dtl.DTLVardef@5f23b3org.highwire.dtl.DTLVardef@20ed2corg.highwire.dtl.DTLVardef@1b3007dorg.highwire.dtl.DTLVardef@e54cf6_HPS_FORMAT_FIGEXP M_FIG C_FIG One sentence summaryY chromosome manipulation yields mostly female mice, preserving litter size without genetic alteration in females. Significance statementThis study describes a genetic approach for producing predominantly female offspring in mice without altering litter size or transmitting genetic modifications to the progeny. A cassette inserted into the Y chromosome of males induces a strong female bias in offspring through a mechanism that remains unclear. The female offspring do not inherit the transgene and are genetically unmodified. This system provides a simple and conditional platform for sex ratio control in mammals, with potential applications in animal breeding and welfare.

The Cell-PCA screen, since its inception, has provided an efficient method for analyzing cellular interactomes and has been used in various biological studies involving proteins like MYC, PER2, and ERK. With rapid advancements in biotechnology, including tools for protein function investigation, the Cell-PCA screen remains relevant. However, despite its successful application in recent studies, there are areas for optimization to ensure its continued relevance in the face of evolving technological advancements.

Widespread treatment of human diseases with gene therapies necessitates the development of gene transfer vectors that integrate genetic information effectively, safely and economically. Accordingly, significant efforts have been devoted to engineer novel tools that i) achieve high-level stable gene transfer at low toxicity to the host cell; ii) induce low levels of genotoxicity and possess a safe integration profile with a high proportion of integrations into safe genomic locations; and iii) are associated with acceptable cost per treatment and scalable/exportable vector production to serve large numbers of patients. The Sleeping Beauty (SB) transposon has been transformed into a vector system that is fulfilling these requirements.\n\nIn the CARAMBA project, we use SB transposition to genetically modify T cells with a chimeric antigen receptor (CAR) specific for the SLAMF7 antigen, that is uniformly and highly expressed on malignant plasma cells in multiple myeloma. We have demonstrated that SLAMF7 CAR-T cells confer specific and very potent anti-myeloma reactivity in pre-clinical models, and are therefore preparing a Phase I/IIa clinical trial of adoptive immunotherapy with autologous, patient-derived SLAMF7-CAR T cells in multiple myeloma (EudraCT Nr. 2019-001264-30/CARAMBA-1).\n\nHere we report on the characterization of genomic safety attributes in SLAMF7 CAR-T cells that we prepared in three clinical-grade manufacturing campaigns under good manufacturing practice (GMP), using T cells that we obtained from three healthy donor volunteers. In the SLAMF7 CAR-T cell product, we determined the average transposon copy number, the genomic insertion profile, and presence of residual SB100X transposase. The data show that the SLAMF7 CAR transposon had been inserted into the T cell genome with the close-to-random distribution pattern that is typical for SB, and with an average transposon copy number ranging between 6 and 12 per T cell. No residual SB100X transposase could be detected by Western blotting in the infusion products. With these attributes, the SLAMF7 CAR-T products satisfy criteria set forth by competent regulatory authorities in order to justify administration of SLAMF7 CAR-T cells to humans in the context of a clinical trial. These data set the stage for the CARAMBA clinical trial, that will be the first in the European Union to use virus-free SB transposition for CAR-T engineering.\n\nDisclosuresThis project is receiving funding from the European Unions Horizon 2020 research and innovation programme under grant agreement No 754658 (CARAMBA).

Recently, the CRISPR-RfxCas13d (CasRx) system was proven to induce efficient mRNA knockdown in animal embryos. Here we compared the efficiency of CasRx-based RNA depletion with that of Cas9-mediated DNA targeting under the same conditions, using automated robotic and manual injection methods. As a proof-of-principle target, we used the no tail (tbxta) gene in zebrafish embryos, for which knockdown and knockout embryonic phenotypes were easy to be scored. Both Cas9 and CasRx systems induced loss of function phenotypes of tbxta gene. Higher percentage of severe phenotype was observed using Cas9 protein compared to the mRNA while the efficiency was similar in terms of Cas13d protein and mRNA. In addition, both the robotic and manual injection approaches yielded similar percentages of phenotypes and mortality rates. Therefore, our study not only showcases the potential of RNA-targeting CRISPR effectors for precise and potent gene knockdown, but also emphasizes automated microinjection in zebrafish embryos as an excellent alternative to manual methods for achieving gene knockdown at a high throughput level.

Crop pests and pathogens annually cause over $100 billion in global crop damage, with insects consuming 5-20% of major grain crops. Current crop pest and disease control strategies rely on insecticidal and fungicidal sprays, plant genetic resistance, transgenes and agricultural practices. dsRNA is emerging as a novel sustainable method of plant protection as an alternative to traditional chemical pesticides. Successful commercialisation of dsRNA based biocontrols requires the economical production of large quantities of dsRNA combined with suitable delivery methods to ensure RNAi efficacy against the target pest. In this study, we have optimised the design of plasmid DNA constructs to produce dsRNA biocontrols in E. coli, by employing a wide range of alternative synthetic transcriptional terminators prior to measurement of dsRNA yield. We demonstrate that a 7.8-fold increase of dsRNA was achieved using triple synthetic transcriptional terminators within a dual T7 dsRNA production system compared to the absence of transcriptional terminators. Moreover, our data demonstrates that batch fermentation production dsRNA using multiple transcriptional terminators is scalable and generates significantly higher yields of dsRNA generated in the absence of transcriptional terminators at both small-scale batch culture and large-scale fermentation. In addition, we show that application of these dsRNA biocontrols expressed in E. coli cells results in increased insect mortality. Finally, novel mass spectrometry analysis was performed to determine the precise sites of transcriptional termination at the different transcriptional terminators providing important further mechanistic insight.

The high efficiency of genome editing presents a challenge when modifying genes associated with viability, welfare, or fertility issues, as implementation of the technology frequently results in mosaic animals with bi-allelic mutations. Combining deactivated Cas9 (dCas9) with Cas9 has been proposed as a strategy to protect one of the two target alleles from editing. We piloted this strategy with 11 genes that are reported as homozygous lethal or associated with welfare issues. We showed that the viability of founders was significantly increased when using 80:20 or 90:10 dCas9:Cas9 ratios, whereas the 70:30 ratio did not yield an equivalent protective effect. The associated overall production rate of mutated founder per manipulated embryo was significantly higher for the 80:20 ratio. Concomitantly, an increased proportion of dCas9 was associated with a significant increase in retention of unedited target alleles but, importantly, did not hinder germline transmission. In addition, editing genes in a paralog cluster with a combination of dCas9 and Cas9 reduced unwanted off-target editing, illustrating a further potential applicability of this approach. This study defines the optimal ratio between dCas9 and Cas9 for strategies aimed at achieving mono-allelic mutations within mosaic founders and proposes a means to reduce the incidence of off-target effects in experiments with limited gRNA options.

Apoptosis is an essential physiological process involved in embryonic development, immune responses, and tissue homeostasis. Despite many studies on pro-apoptotic genes, few reports have directly compared the lethality-inducing potential between them under identical conditions. In this study, we evaluated the lethality-inducing potential of three representative pro-apoptotic genes, Bax, Casp3, and Casp9, in mouse early embryos under defined conditions using the doxycycline (Dox)-inducible tetracycline-regulated gene expression system (Tet-On system) in combination with the PiggyBac transposon system. All genes were transcriptionally induced by Dox, and Bax showed the strongest lethal effect, followed by Casp9, while Casp3 did not show any effect. Notably, Bax expression severely impaired blastocyst formation and led to the intense accumulation of the DNA damage marker {gamma}H2AX. These findings suggest that introducing upstream apoptotic regulators leads to the more efficient and widespread activation of the apoptotic cascade. Additionally, an unexpected Dox-dependent increase in the expression of reverse tetracycline-controlled transactivator, which is typically driven by a constitutive promoter, was observed, raising the possibility of unanticipated regulatory mechanisms within the Tet-On system. Overall, this study is expected to contribute to a deeper understanding of apoptotic mechanisms and future advancements in regenerative medicine, reproductive engineering, and cancer research.

Genetic biocontrol interventions targeting mosquito-borne diseases require the release of male mosquitoes exclusively, as only females consume blood and transmit human pathogens. This reduces the risk of spreading pathogens while enabling effective population control. Robust sex sorting methods to enable early larval sorting in mosquitoes need to be developed to allow for scalable sex sorting for genetic biocontrol interventions. This study applies the SEPARATOR (Sexing Element Produced by Alternative RNA-splicing of A Transgenic Observable Reporter) system, previously developed for Aedes aegypti, to the Asian malaria vector Anopheles stephensi. We hypothesized that the intron from the doublesex gene in Anopheles gambiae would function in An. stephensi due to evolutionary conservation. Our results confirm that the splicing module from An. gambiae operates effectively in An. stephensi, demonstrating evolutionary conservation in sex-specific splicing events between these species. This system enables reliable positive male selection from first instar larval to pupal stages. RT-PCR analysis demonstrates that male-specific EGFP expression is dependent on doublesex sex-specific splicing events. The SEPARATOR systems independence from sex-chromosome linkage confers resistance to meiotic recombination and chromosomal rearrangements. This approach may facilitate the mass release of males, and the cross-species portability of SEPARATOR establishes it as a valuable tool for genetic biocontrol interventions across various pest species.

Electroporation is the most feasible non-viral material delivery system for manipulating human T cells given its time- and cost-effectiveness. However, efficient delivery requires electroporation settings to be optimized for different devices, cellular states, and materials to be delivered. Here, we used electroporation to either induce exogenous gene expression in human primary T cells by plasmids or in vitro transcribed (IVT) mRNA and also target endogenous genes by Cas9 ribonucleoproteins (RNPs). We characterized the electroporation conditions both for activated and unstimulated human T cells. Although naive cells are non-dividing and therefore their genetic manipulation is harder compared to activated T cells, we developed the technical ability to manipulate both naive and memory cells within the unstimulated T cell population by IVT mRNA and Cas9 RNP electroporation. Here, we outline the best practices for achieving highly-efficient genetic manipulation in primary T cells without causing significant cytotoxicity to the cells. Because there is increasing evidence for \"less-differentiated\" T cells to have better anti-tumor activity for immunotherapy, manipulating naive T cells with high efficiency is also of high importance to clinical applications and to study the biology of these cells.

Plasmid vectors are well established tools used to genetically engineer bacteria both in the laboratory and at industrial scale. The past few decades have seen a rising interest in the use of plasmid DNA (pDNA) for biotherapeutic applications. This interest is a strong driver for the development of technologies to increase pDNA production at biopharmaceutical scale in terms of decreasing production costs and meeting regulatory requirements. Although cell free technologies are emerging, pDNA vectors are still produced by fermentation in Escherichia coli strains. As plasmids are extra-chromosomic molecules there is a probability of losing a certain ratio within the E. coli population during the fermentation process leading to a decrease of DNA production efficiency. Maintaining pDNA in the population is thus a key element to reach efficient and robust production. Traditionally, antibiotic resistance genes and antibiotics have been used to generate a selective pressure to ensure pDNA stability in the microbial population during the production process. Nowadays, having an antibiotic resistance gene in the pDNA coding sequence represents a limitation both for safety and legal requirements and in terms of production yield. For this reason, we have developed a pDNA antibiotic-free bacteriocin-based selection system, based on the genes involved in the production, processing, secretion and immunity of the bacteriocin microcin V. Our approach is based on the peer pressure exerted by the bacteriocin and does not rely on the addition of any selective agent in the medium to limit population drift and ensure plasmid stability. This novel antibiotic-free approach may be applied to any pDNA vector in different E. coli strains and expands their potential applications in both animal and human health as delivery vectors for biotherapeutics.