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1.
Methods Mol Biol ; 2829: 109-126, 2024.
Article in English | MEDLINE | ID: mdl-38951330

ABSTRACT

Baculoviruses are widely used for their potential as biological pesticide and as platform for the production of recombinant proteins and gene therapy vectors. The Baculovirus Expression Vector System (BEVS) is used for high level of expression of (multiple) proteins in insect cells. Baculovirus recombinants can be quickly constructed by transposition of the gene(s) of interest into a so-called bacmid, which is a baculovirus infectious clone maintained as single-copy, bacterial artificial chromosome in Escherichia coli. A two-step homologous recombineering technique using the lambda-red system in E. coli allows for scarless editing of the bacmid with PCR products based on sequence homology. In the first step, a selection cassette with 50 bp homology arms, typically generated by PCR, is inserted into the designated locus. In the second step, the selection cassette is removed based on a negative selection marker, such as SacB or rpsL. This lambda-red recombineering technique can be used for multiple gene editing purposes, including (large) deletions, insertions, and even single point mutations. Moreover, since there are no remnants of the editing process, successive modifications of the same bacmid are possible. This chapter provides detailed instructions to design and perform two-step homologous recombineering of baculovirus bacmid DNA in E. coli. We present two case studies demonstrating the utility of this technique for creating a deletion mutant of the chitinase and cathepsin genes and for introducing a single point mutation in the baculovirus gene gp41. This scarless genome editing approach can facilitate functional studies of baculovirus genes and improve the production of recombinant proteins using the BEVS.


Subject(s)
Baculoviridae , Escherichia coli , Gene Editing , Genetic Vectors , Gene Editing/methods , Escherichia coli/genetics , Baculoviridae/genetics , Genetic Vectors/genetics , Chromosomes, Artificial, Bacterial/genetics , Genome, Viral , Genetic Engineering/methods , Bacteriophage lambda/genetics , Homologous Recombination
2.
World J Microbiol Biotechnol ; 40(9): 263, 2024 Jul 09.
Article in English | MEDLINE | ID: mdl-38980547

ABSTRACT

Genetically engineered cyanobacterial strains that have improved growth rate, biomass productivity, and metabolite productivity could be a better option for sustainable bio-metabolite production. The global demand for biobased metabolites with nutraceuticals and health benefits has increased due to their safety and plausible therapeutic and nutritional utility. Cyanobacteria are solar-powered green cellular factories that can be genetically tuned to produce metabolites with nutraceutical and pharmaceutical benefits. The present review discusses biotechnological endeavors for producing bioprospective compounds from genetically engineered cyanobacteria and discusses the challenges and troubleshooting faced during metabolite production. This review explores the cyanobacterial versatility, the use of engineered strains, and the techno-economic challenges associated with scaling up metabolite production from cyanobacteria. Challenges to produce cyanobacterial bioactive compounds with remarkable nutraceutical values have been discussed. Additionally, this review also summarises the challenges and future prospects of metabolite production from genetically engineered cyanobacteria as a sustainable approach.


Subject(s)
Biotechnology , Cyanobacteria , Dietary Supplements , Metabolic Engineering , Cyanobacteria/genetics , Cyanobacteria/metabolism , Metabolic Engineering/methods , Biotechnology/methods , Genetic Engineering , Biomass
3.
J Environ Manage ; 365: 121707, 2024 Aug.
Article in English | MEDLINE | ID: mdl-38968883

ABSTRACT

Addressing the threat of harmful cyanobacterial blooms (CyanoHABs) and their associated microcystins (MCs) is crucial for global drinking water safety. In this review, we comprehensively analyze and compares the physical, chemical, and biological methods and genetic engineering for MCs degradation in aquatic environments. Physical methods, such as UV treatments and photocatalytic reactions, have a high efficiency in breaking down MCs, with the potential for further enhancement in performance and reduction of hazardous byproducts. Chemical treatments using chlorine dioxide and potassium permanganate can reduce MC levels but require careful dosage management to avoid toxic by-products and protect aquatic ecosystems. Biological methods, including microbial degradation and phytoremediation techniques, show promise for the biodegradation of MCs, offering reduced environmental impact and increased sustainability. Genetic engineering, such as immobilization of microcystinase A (MlrA) in Escherichia coli and its expression in Synechocystis sp., has proven effective in decomposing MCs such as MC-LR. However, challenges related to specific environmental conditions such as temperature variations, pH levels, presence of other contaminants, nutrient availability, oxygen levels, and light exposure, as well as scalability of biological systems, necessitate further exploration. We provide a comprehensive evaluation of MCs degradation techniques, delving into their practicality, assessing the environmental impacts, and scrutinizing their efficiency to offer crucial insights into the multifaceted nature of these methods in various environmental contexts. The integration of various methodologies to enhance degradation efficiency is vital in the field of water safety, underscoring the need for ongoing innovation.


Subject(s)
Biodegradation, Environmental , Genetic Engineering , Microcystins , Microcystins/metabolism , Cyanobacteria/metabolism
4.
Front Immunol ; 15: 1411393, 2024.
Article in English | MEDLINE | ID: mdl-38962002

ABSTRACT

Chimeric antigen receptor (CAR) T-cell therapy has proven a breakthrough in cancer treatment in the last decade, giving unprecedented results against hematological malignancies. All approved CAR T-cell products, as well as many being assessed in clinical trials, are generated using viral vectors to deploy the exogenous genetic material into T-cells. Viral vectors have a long-standing clinical history in gene delivery, and thus underwent iterations of optimization to improve their efficiency and safety. Nonetheless, their capacity to integrate semi-randomly into the host genome makes them potentially oncogenic via insertional mutagenesis and dysregulation of key cellular genes. Secondary cancers following CAR T-cell administration appear to be a rare adverse event. However several cases documented in the last few years put the spotlight on this issue, which might have been underestimated so far, given the relatively recent deployment of CAR T-cell therapies. Furthermore, the initial successes obtained in hematological malignancies have not yet been replicated in solid tumors. It is now clear that further enhancements are needed to allow CAR T-cells to increase long-term persistence, overcome exhaustion and cope with the immunosuppressive tumor microenvironment. To this aim, a variety of genomic engineering strategies are under evaluation, most relying on CRISPR/Cas9 or other gene editing technologies. These approaches are liable to introduce unintended, irreversible genomic alterations in the product cells. In the first part of this review, we will discuss the viral and non-viral approaches used for the generation of CAR T-cells, whereas in the second part we will focus on gene editing and non-gene editing T-cell engineering, with particular regard to advantages, limitations, and safety. Finally, we will critically analyze the different gene deployment and genomic engineering combinations, delineating strategies with a superior safety profile for the production of next-generation CAR T-cell.


Subject(s)
Gene Editing , Immunotherapy, Adoptive , Receptors, Chimeric Antigen , T-Lymphocytes , Humans , Immunotherapy, Adoptive/methods , Immunotherapy, Adoptive/adverse effects , Receptors, Chimeric Antigen/genetics , Receptors, Chimeric Antigen/immunology , Gene Editing/methods , T-Lymphocytes/immunology , Animals , Neoplasms/therapy , Neoplasms/immunology , Neoplasms/genetics , Genetic Vectors/genetics , Genetic Vectors/immunology , Genetic Engineering , CRISPR-Cas Systems , Receptors, Antigen, T-Cell/genetics , Receptors, Antigen, T-Cell/immunology , Tumor Microenvironment/immunology
5.
J Immunother Cancer ; 12(7)2024 Jul 04.
Article in English | MEDLINE | ID: mdl-38964783

ABSTRACT

BACKGROUND: T cells play a central role in the antitumor response. However, they often face numerous hurdles in the tumor microenvironment, including the scarcity of available essential metabolites such as glucose and amino acids. Moreover, cancer cells can monopolize these resources to thrive and proliferate by upregulating metabolite transporters and maintaining a high metabolic rate, thereby outcompeting T cells. METHODS: Herein, we sought to improve T-cell antitumor function in the tumor vicinity by enhancing their glycolytic capacity to better compete with tumor cells. To achieve this, we engineered human T cells to express a key glycolysis enzyme, phosphofructokinase, in conjunction with Glucose transporter 3, a glucose transporter. We co-expressed these, along with tumor-specific chimeric antigen or T-cell receptors. RESULTS: Engineered cells demonstrated an increased cytokine secretion and upregulation of T-cell activation markers compared with control cells. Moreover, they displayed superior glycolytic capacity, which translated into an improved in vivo therapeutic potential in a xenograft model of human tumors. CONCLUSION: In summary, these findings support the implementation of T-cell metabolic engineering to enhance the efficacy of cellular immunotherapies for cancer.


Subject(s)
Glycolysis , T-Lymphocytes , Humans , Animals , T-Lymphocytes/immunology , T-Lymphocytes/metabolism , Mice , Genetic Engineering , Tumor Microenvironment , Cell Line, Tumor , Neoplasms/immunology , Neoplasms/metabolism , Xenograft Model Antitumor Assays
6.
Annu Rev Anal Chem (Palo Alto Calif) ; 17(1): 393-410, 2024 Jul.
Article in English | MEDLINE | ID: mdl-39018352

ABSTRACT

Bacteriophages, which are viral predators of bacteria, have evolved to efficiently recognize, bind, infect, and lyse their host, resulting in the release of tens to hundreds of propagated viruses. These abilities have attracted biosensor developers who have developed new methods to detect bacteria. Recently, several comprehensive reviews have covered many of the advances made regarding the performance of phage-based biosensors. Therefore, in this review, we first describe the landscape of phage-based biosensors and then cover advances in other aspects of phage biology and engineering that can be used to make high-impact contributions to biosensor development. Many of these advances are in fields adjacent to analytical chemistry such as synthetic biology, machine learning, and genetic engineering and will allow those looking to develop phage-based biosensors to start taking alternative approaches, such as a bottom-up design and synthesis of custom phages with the singular task of detecting their host.


Subject(s)
Bacteriophages , Biosensing Techniques , Bacteriophages/chemistry , Biosensing Techniques/methods , Bacteria/virology , Genetic Engineering
7.
Methods Mol Biol ; 2819: 157-187, 2024.
Article in English | MEDLINE | ID: mdl-39028507

ABSTRACT

The development of novel DNA assembly methods in recent years has paved the way for the construction of synthetic replicons to be used for basic research and biotechnological applications. A learning-by-building approach can now answer questions about how chromosomes must be constructed to maintain genetic information. Here we describe an efficient pipeline for the design and assembly of synthetic, secondary chromosomes in Escherichia coli based on the popular modular cloning (MoClo) system.


Subject(s)
Escherichia coli , Synthetic Biology , Escherichia coli/genetics , Synthetic Biology/methods , Cloning, Molecular/methods , Genetic Engineering/methods , Replicon/genetics , Chromosomes, Bacterial/genetics , Plasmids/genetics , Chromosomes/genetics
8.
Methods Mol Biol ; 2819: 241-260, 2024.
Article in English | MEDLINE | ID: mdl-39028510

ABSTRACT

Bacillus subtilis is one of the best-studied bacteria and serves as a Gram-positive model system to address fundamental biological processes. Depending on conditions, a B. subtilis cell can initiate one out of various distinct differentiation processes to cope with changing environmental conditions. One of these differentiation processes is natural competence that allows cells to adsorb exogenous DNA and subsequently incorporate it into its chromosome by homologous recombination. Due to competence development, the genome of B. subtilis can be easily manipulated, and this has contributed to B. subtilis being a model system. In this chapter, we describe some of the most common genetic tools that can be used in combination with natural competence to tailor the genome of B. subtilis.


Subject(s)
Bacillus subtilis , Genetic Engineering , Homologous Recombination , Bacillus subtilis/genetics , Genetic Engineering/methods , Genome, Bacterial
9.
ACS Synth Biol ; 13(7): 2227-2237, 2024 Jul 19.
Article in English | MEDLINE | ID: mdl-38975718

ABSTRACT

The inevitable transition from petrochemical production processes to renewable alternatives has sparked the emergence of biofoundries in recent years. Manual engineering of microbes will not be sufficient to meet the ever-increasing demand for novel producer strains. Here we describe the AutoBioTech platform, a fully automated laboratory system with 14 devices to perform operations for strain construction without human interaction. Using modular workflows, this platform enables automated transformations of Escherichia coli with plasmids assembled via modular cloning. A CRISPR/Cas9 toolbox compatible with existing modular cloning frameworks allows automated and flexible genome editing of E. coli. In addition, novel workflows have been established for the fully automated transformation of the Gram-positive model organism Corynebacterium glutamicum by conjugation and electroporation, with the latter proving to be the more robust technique. Overall, the AutoBioTech platform excels at versatility due to the modularity of workflows and seamless transitions between modules. This will accelerate strain engineering of Gram-negative and Gram-positive bacteria.


Subject(s)
CRISPR-Cas Systems , Corynebacterium glutamicum , Escherichia coli , Gene Editing , Plasmids , Escherichia coli/genetics , Corynebacterium glutamicum/genetics , Corynebacterium glutamicum/metabolism , Plasmids/genetics , Gene Editing/methods , CRISPR-Cas Systems/genetics , Electroporation/methods , Genetic Engineering/methods
10.
J Zhejiang Univ Sci B ; 25(7): 557-567, 2024 Jul 11.
Article in English, Chinese | MEDLINE | ID: mdl-39011676

ABSTRACT

Cancer immunotherapy has rapidly become the fourth mainstream treatment alternative after surgery, radiotherapy, and chemotherapy, with some promising results. It aims to kill tumor cells by mobilizing or stimulating cytotoxic immune cells. However, the clinical applications of tumor immunotherapies are limited owing to a lack of adequate delivery pathways and high toxicity. Recently, nanomaterials and genetic engineering have shown great potential in overcoming these limitations by protecting the delivery of antigens, activating targeted T cells, modulating the immunosuppressive tumor microenvironment, and improving the treatment efficacy. Bacillus Calmette-Guérin (BCG) is a live attenuated Mycobacterium bovis vaccine used to prevent tuberculosis, which was first reported to have antitumor activity in 1927. BCG therapy can activate the immune system by inducing various cytokines and chemokines, and its specific immune and inflammatory responses exert antitumor effects. BCG was first used during the 1970s as an intravesical treatment agent for bladder cancer, which effectively improved immune antitumor activity and prevented tumor recurrence. More recently, nano-BCG and genetically engineered BCG have been proposed as treatment alternatives for bladder cancer due to their ability to induce stronger and more stable immune responses. In this study, we outline the development of nano-BCG and genetically engineered BCG for bladder cancer immunotherapy and review their potential and associated challenges.


Subject(s)
BCG Vaccine , Immunotherapy , Urinary Bladder Neoplasms , Urinary Bladder Neoplasms/therapy , Urinary Bladder Neoplasms/immunology , Humans , Immunotherapy/methods , BCG Vaccine/therapeutic use , Animals , Tumor Microenvironment , Nanoparticles , Mycobacterium bovis , Genetic Engineering
11.
J Biosci Bioeng ; 138(2): 105-110, 2024 Aug.
Article in English | MEDLINE | ID: mdl-38825559

ABSTRACT

Omega-3 polyunsaturated fatty acids (PUFAs), such as eicosapentaenoic acid (EPA; C20:5n-3) and docosahexaenoic acid (DHA; C22:6n-3) are widely used as additives in fish feed in the aquaculture sector. To date, the supply of omega-3 PUFAs have heavily depended upon fish oil production. As the need for omega-3 PUFAs supply for the growing population increases, a more sustainable approach is required to keep up with the demand. The oleaginous diatom Fistulifera solaris is known to synthesize EPA with the highest level among autotrophically cultured microalgae, however, this species does not accumulate significant amounts of DHA, which, in some cases, is required in aquaculture rather than EPA. This is likely due to the lack of expression of essential enzymes namely Δ5 elongase (Δ5ELO) and Δ4 desaturase. In this study, we identified endogenous Δ5ELO genes in F. solaris and introduced recombinant expression cassettes harboring Δ5ELO into F. solaris through bacterial conjugation. As a result, it managed to induce the synthesis of docosapentaenoic acid (DPA; C22:5n-3), a direct precursor of DHA. This study paves the way for expanding our understanding of the omega-3 PUFAs pathway using endogenous genes in the oleaginous diatom.


Subject(s)
Diatoms , Docosahexaenoic Acids , Eicosapentaenoic Acid , Fatty Acids, Omega-3 , Diatoms/metabolism , Diatoms/genetics , Fatty Acids, Omega-3/metabolism , Eicosapentaenoic Acid/metabolism , Eicosapentaenoic Acid/biosynthesis , Docosahexaenoic Acids/metabolism , Docosahexaenoic Acids/biosynthesis , Fatty Acid Desaturases/metabolism , Fatty Acid Desaturases/genetics , Genetic Engineering , Fatty Acid Elongases/metabolism , Fatty Acid Elongases/genetics , Microalgae/metabolism , Microalgae/genetics , Aquaculture
12.
Nat Commun ; 15(1): 4896, 2024 Jun 08.
Article in English | MEDLINE | ID: mdl-38851790

ABSTRACT

Biological computing is a promising field with potential applications in biosafety, environmental monitoring, and personalized medicine. Here we present work on the design of bacterial computers using spatial patterning to process information in the form of diffusible morphogen-like signals. We demonstrate, mathematically and experimentally, that single, modular, colonies can perform simple digital logic, and that complex functions can be built by combining multiple colonies, removing the need for further genetic engineering. We extend our experimental system to incorporate sender colonies as morphogen sources, demonstrating how one might integrate different biochemical inputs. Our approach will open up ways to perform biological computation, with applications in bioengineering, biomaterials and biosensing. Ultimately, these computational bacterial communities will help us explore information processing in natural biological systems.


Subject(s)
Escherichia coli , Escherichia coli/metabolism , Escherichia coli/genetics , Bacteria/metabolism , Bacteria/genetics , Genetic Engineering/methods , Diffusion , Models, Biological , Bioengineering/methods
13.
Front Immunol ; 15: 1404668, 2024.
Article in English | MEDLINE | ID: mdl-38903492

ABSTRACT

Heart transplantation is associated with major hurdles, including the limited number of available organs for transplantation, the risk of rejection due to genetic discrepancies, and the burden of immunosuppression. In this study, we demonstrated the feasibility of permanent genetic engineering of the heart during ex vivo perfusion. Lentiviral vectors encoding for short hairpin RNAs targeting beta2-microglobulin (shß2m) and class II transactivator (shCIITA) were delivered to the graft during two hours of normothermic EVHP. Highly efficient genetic engineering was indicated by stable reporter gene expression in endothelial cells and cardiomyocytes. Remarkably, swine leucocyte antigen (SLA) class I and SLA class II expression levels were decreased by 66% and 76%, respectively, in the vascular endothelium. Evaluation of lactate, troponin T, and LDH levels in the perfusate and histological analysis showed no additional cell injury or tissue damage caused by lentiviral vectors. Moreover, cytokine secretion profiles (IL-6, IL-8, and TNF-α) of non-transduced and lentiviral vector-transduced hearts were comparable. This study demonstrated the ex vivo generation of genetically engineered hearts without compromising tissue integrity. Downregulation of SLA expression may contribute to reduce the immunogenicity of the heart and support graft survival after allogeneic or xenogeneic transplantation.


Subject(s)
Genetic Vectors , Heart Transplantation , Histocompatibility Antigens Class I , Lentivirus , Animals , Lentivirus/genetics , Heart Transplantation/methods , Genetic Vectors/genetics , Swine , Histocompatibility Antigens Class I/genetics , Histocompatibility Antigens Class I/immunology , Histocompatibility Antigens Class I/metabolism , Perfusion/methods , Histocompatibility Antigens Class II/genetics , Histocompatibility Antigens Class II/metabolism , Histocompatibility Antigens Class II/immunology , beta 2-Microglobulin/genetics , Cytokines/metabolism , Genetic Engineering , Myocytes, Cardiac/metabolism , Myocytes, Cardiac/immunology , Humans , RNA, Small Interfering/genetics , Graft Survival/immunology , Graft Survival/genetics , Endothelial Cells/metabolism , Endothelial Cells/immunology , Nuclear Proteins , Trans-Activators
14.
Sci Rep ; 14(1): 13657, 2024 06 13.
Article in English | MEDLINE | ID: mdl-38871942

ABSTRACT

This work aimed to design a synthetic salt-inducible promoter using a cis-engineering approach. The designed promoter (PS) comprises a minimal promoter sequence for basal-level expression and upstream cis-regulatory elements (CREs) from promoters of salinity-stress-induced genes. The copy number, spacer lengths, and locations of CREs were manually determined based on their occurrence within native promoters. The initial activity profile of the synthesized PS promoter in transiently transformed N. tabacum leaves shows a seven-fold, five-fold, and four-fold increase in reporter GUS activity under salt, drought, and abscisic acid stress, respectively, at the 24-h interval, compared to the constitutive CaMV35S promoter. Analysis of gus expression in stable Arabidopsis transformants showed that the PS promoter induces over a two-fold increase in expression under drought or abscisic acid stress and a five-fold increase under salt stress at 24- and 48-h intervals, compared to the CaMV35S promoter. The promoter PS exhibits higher and more sustained activity under salt, drought, and abscisic acid stress compared to the constitutive CaMV35S.


Subject(s)
Abscisic Acid , Arabidopsis , Gene Expression Regulation, Plant , Promoter Regions, Genetic , Arabidopsis/genetics , Abscisic Acid/pharmacology , Plants, Genetically Modified/genetics , Droughts , Nicotiana/genetics , Stress, Physiological/genetics , Sodium Chloride/pharmacology , Genetic Engineering/methods , Salt Stress/genetics
15.
Appl Microbiol Biotechnol ; 108(1): 378, 2024 Jun 18.
Article in English | MEDLINE | ID: mdl-38888816

ABSTRACT

Non-symbiotic N2-fixation would greatly increase the versatility of N-biofertilizers for sustainable agriculture. Genetic modification of diazotrophic bacteria has successfully enhanced NH4+ release. In this study, we compared the competitive fitness of A. vinelandii mutant strains, which allowed us to analyze the burden of NH4+ release under a broad dynamic range. Long-term competition assays under regular culture conditions confirmed a large burden for NH4+ release, exclusion by the wt strain, phenotypic instability, and loss of the ability to release NH4+. In contrast, co-inoculation in mild autoclaved soil showed a much longer co-existence with the wt strain and a stable NH4+ release phenotype. All genetically modified strains increased the N content and changed its chemical speciation in the soil. This study contributes one step forward towards bridging a knowledge gap between molecular biology laboratory research and the incorporation of N from the air into the soil in a molecular species suitable for plant nutrition, a crucial requirement for developing improved bacterial inoculants for economic and environmentally sustainable agriculture. KEY POINTS: • Genetic engineering for NH4+ excretion imposes a fitness burden on the culture medium • Large phenotypic instability for NH4+-excreting bacteria in culture medium • Lower fitness burden and phenotypic instability for NH4+-excreting bacteria in soil.


Subject(s)
Ammonium Compounds , Azotobacter vinelandii , Soil Microbiology , Azotobacter vinelandii/genetics , Azotobacter vinelandii/metabolism , Ammonium Compounds/metabolism , Nitrogen Fixation , Nitrogen/metabolism , Genetic Fitness , Phenotype , Soil/chemistry , Culture Media/chemistry , Genetic Engineering
16.
Nat Commun ; 15(1): 4924, 2024 Jun 10.
Article in English | MEDLINE | ID: mdl-38858354

ABSTRACT

Targeted gene delivery to the brain is a critical tool for neuroscience research and has significant potential to treat human disease. However, the site-specific delivery of common gene vectors such as adeno-associated viruses (AAVs) is typically performed via invasive injections, which limit its applicable scope of research and clinical applications. Alternatively, focused ultrasound blood-brain-barrier opening (FUS-BBBO), performed noninvasively, enables the site-specific entry of AAVs into the brain from systemic circulation. However, when used in conjunction with natural AAV serotypes, this approach has limited transduction efficiency and results in substantial undesirable transduction of peripheral organs. Here, we use high throughput in vivo selection to engineer new AAV vectors specifically designed for local neuronal transduction at the site of FUS-BBBO. The resulting vectors substantially enhance ultrasound-targeted gene delivery and neuronal tropism while reducing peripheral transduction, providing a more than ten-fold improvement in targeting specificity in two tested mouse strains. In addition to enhancing the only known approach to noninvasively target gene delivery to specific brain regions, these results establish the ability of AAV vectors to be evolved for specific physical delivery mechanisms.


Subject(s)
Blood-Brain Barrier , Brain , Dependovirus , Gene Transfer Techniques , Genetic Vectors , Animals , Genetic Vectors/genetics , Genetic Vectors/administration & dosage , Dependovirus/genetics , Mice , Blood-Brain Barrier/metabolism , Brain/metabolism , Humans , Neurons/metabolism , Transduction, Genetic/methods , Mice, Inbred C57BL , Genetic Engineering/methods , Female , Male , HEK293 Cells
17.
Int J Biol Macromol ; 271(Pt 2): 132696, 2024 Jun.
Article in English | MEDLINE | ID: mdl-38823737

ABSTRACT

Lignin is a complex polymer found in the cell walls of plants, providing structural support and protection against pathogens. By modifying lignin composition and structure, scientists aim to optimize plant defense responses and increase resistance to pathogens. This can be achieved through various genetic engineering techniques which involve manipulating the genes responsible for lignin synthesis. By either up regulating or down regulating specific genes, researchers can alter the lignin content, composition, or distribution in plant tissues. Reducing lignin content in specific tissues like leaves can improve the effectiveness of defense mechanisms by allowing for better penetration of antimicrobial compounds. Overall, Lignin modification through techniques has shown promising results in enhancing various plants resistance against pathogens. Furthermore, lignin modification can have additional benefits beyond pathogen resistance. It can improve biomass processing for biofuel production by reducing lignin recalcitrance, making the extraction of sugars from cellulose more efficient. The complexity of lignin biosynthesis and its interactions with other plant components make it a challenging target for modification. Additionally, the potential environmental impact and regulatory considerations associated with genetically modified organisms (GMOs) require careful evaluation. Ongoing research aims to further optimize this approach and develop sustainable solutions for crop protection.


Subject(s)
Lignin , Plant Diseases , Lignin/metabolism , Lignin/chemistry , Plant Diseases/genetics , Plant Diseases/microbiology , Plant Diseases/prevention & control , Disease Resistance/genetics , Plants, Genetically Modified , Plants/metabolism , Plants/genetics , Genetic Engineering , Biomass
19.
Methods Mol Biol ; 2822: 419-429, 2024.
Article in English | MEDLINE | ID: mdl-38907932

ABSTRACT

Ribozymes engineered from the RNase P catalytic RNA (M1 RNA) represent promising gene-targeting agents for clinical applications. We describe in this report an in vitro amplification and selection procedure for generating active RNase P ribozyme variants with improved catalytic efficiency. Using the amplification and selection procedure, we have previously generated ribozyme variants that were highly active in cleaving a herpes simplex virus 1-encoded mRNA in vitro and inhibiting its expression in virally infected human cells. In this chapter, we use an overlapping region of the mRNAs for the IE1 and IE2 proteins of human cytomegalovirus (HCMV) as a target substrate. We provide detailed protocols and include methods for establishing the procedure for the amplification and selection of active mRNA-cleaving RNase P ribozymes. The in vitro amplification and selection system represents an excellent approach for engineering highly active RNase P ribozymes that can be used in both basic research and clinical applications.


Subject(s)
Gene Targeting , RNA, Catalytic , Ribonuclease P , Ribonuclease P/genetics , Ribonuclease P/metabolism , RNA, Catalytic/genetics , RNA, Catalytic/metabolism , Humans , Gene Targeting/methods , RNA, Messenger/genetics , RNA, Messenger/metabolism , Genetic Engineering/methods , Cytomegalovirus/genetics
20.
Nat Commun ; 15(1): 5319, 2024 Jun 22.
Article in English | MEDLINE | ID: mdl-38909033

ABSTRACT

Although CRISPR-dCas13, the RNA-guided RNA-binding protein, was recently exploited as a translation-level gene expression modulator, it has still been difficult to precisely control the level due to the lack of detailed characterization. Here, we develop a synthetic tunable translation-level CRISPR interference (Tl-CRISPRi) system based on the engineered guide RNAs that enable precise and predictable down-regulation of mRNA translation. First, we optimize the Tl-CRISPRi system for specific and multiplexed repression of genes at the translation level. We also show that the Tl-CRISPRi system is more suitable for independently regulating each gene in a polycistronic operon than the transcription-level CRISPRi (Tx-CRISPRi) system. We further engineer the handle structure of guide RNA for tunable and predictable repression of various genes in Escherichia coli and Vibrio natriegens. This tunable Tl-CRISPRi system is applied to increase the production of 3-hydroxypropionic acid (3-HP) by 14.2-fold via redirecting the metabolic flux, indicating the usefulness of this system for the flux optimization in the microbial cell factories based on the RNA-targeting machinery.


Subject(s)
CRISPR-Cas Systems , Escherichia coli , Protein Biosynthesis , RNA, Guide, CRISPR-Cas Systems , Vibrio , Escherichia coli/genetics , Escherichia coli/metabolism , RNA, Guide, CRISPR-Cas Systems/genetics , RNA, Guide, CRISPR-Cas Systems/metabolism , Vibrio/genetics , Vibrio/metabolism , Gene Expression Regulation, Bacterial , RNA, Messenger/genetics , RNA, Messenger/metabolism , Clustered Regularly Interspaced Short Palindromic Repeats/genetics , Operon/genetics , Genetic Engineering/methods , Lactic Acid/analogs & derivatives
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