Genetic Engineering Tools Research Articles

Macroalgae: Marine players in vaccinology

Although seaweeds have been used for decades as food, medicinal plants, and nutritional supplements, their use as a vaccine production and delivery platform has been barely explored, despite the availability of several genetic tools for seaweed transformation. The present review introduces seaweeds and their valuable immunomodulatory compounds in perspective as vaccine production and mucosal delivery platform. Genetic engineering tools are reviewed for macroalgal transformation, and the recombinant proteins produced in this platform are summarized. Finally, the hurdles and opportunities on seaweed potential as a vaccine production platform and delivery vehicle are proposed. As expected, seaweeds can soon be exploited to produce vaccine candidates due to their biological characteristics, including accelerated growth, photosynthetic capacity, and bioactive compound production, as well as the genetic engineering tools available. With all these properties -plus the commercial seaweed industry- this new envisioned technology can be adopted to improve human and animal health.

Vibrio natriegens: Application of a Fast-Growing Halophilic Bacterium.

The fast growth accompanied with high substrate consumption rates and a versatile metabolism paved the way to exploit Vibrio natriegens as unconventional host for biotechnological applications. Meanwhile, a wealth of knowledge on the physiology, the metabolism, and the regulation in this halophilic marine bacterium has been gathered. Sophisticated genetic engineering tools and metabolic models are available and have been applied to engineer production strains and first chassis variants of V. natriegens. In this review, we update the current knowledge on the physiology and the progress in the development of synthetic biology tools and provide an overview of recent advances in metabolic engineering of this promising host. We further discuss future challenges to enhance the application range of V. natriegens.

From classical approaches to new developments in genetic engineering of live attenuated vaccine against cutaneous leishmaniasis: potential and immunization.

Despite the development of a vaccine against cutaneous leishmaniasis in preclinical and clinical studies, we still do not have a safe and effective vaccine for human use. Given this situation, the search for a new prophylactic alternative to control leishmaniasis should be a global priority. A first-generation vaccine strategy-leishmanization, in which live Leishmania major parasites are inoculated into the skin to protect against reinfection, is taking advantage of this situation. Live attenuated Leishmania vaccine candidates are promising alternatives due to their robust protective immune responses. Importantly, they do not cause disease and could provide long-term protection following challenges with a virulent strain. In addition to physical and chemical methods, genetic tools, including the Cre-loxP system, have enabled the selection of safer null mutant live attenuated Leishmania parasites obtained by gene disruption. This was followed by the discovery and introduction of CRISPR/Cas-based gene editing tools, which can be easily and precisely used to modify genes. Here, we briefly review the immunopathology of L. major parasites and then present the classical methods and their limitations for the production of live attenuated vaccines. We then discuss the potential of current genetic engineering tools to generate live attenuated vaccine strains by targeting key genes involved in L. major pathogenesis and then discuss their discovery and implications for immune responses to control leishmaniasis.

Application prospect of CRISPR-Cas9 gene editing in epilepsy

Epilepsy, a chronic noncommunicable brain disorder, is characterized by abnormal electrical activity in the brain, leading to seizures and disruptions in normal brain functions. Despite various known causes, a significant proportion of epilepsy cases remain unexplained. In recent years, Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR-Cas9) technology has emerged as a powerful tool for genetic engineering. Compared with the first two generations of gene-editing technology, it has the advantages of low cost, easy design and easy operation. Utilizing CRISPR activation (CRISPRa), researchers have explored the potential of increasing the expression of genes involved in regulating synaptic interactions to control epileptic activity. Studies on transgenic mice have shown that upregulating the Kv1.1 gene (Kcnal1), which encodes for a voltage-gated potassium channel responsible for regulating neuronal excitability, can reduce seizures and improve cognitive function. Additionally, CRISPR-Cas9 has been instrumental in creating animal models to study epilepsy, providing insights into gene functions, disease mechanisms, and potential therapeutic interventions. However, further research is needed to fully explore the potential of CRISPR-based therapies for targeted treatment of epilepsy. This review systematically introduces the pathogenesis of epilepsy, including the origins and causes of epilepsy and the mechanism of seizure formation and further discusses the application of the CRISPR/Cas9 system in epilepsy.

Precise genetic engineering with piggyBac transposon in plants.

Transposons are mobile genetic elements that can move to a different position within a genome or between genomes. They have long been used as a tool for genetic engineering, including transgenesis, insertional mutagenesis, and marker excision, in a variety of organisms. The piggyBac transposon derived from the cabbage looper moth is one of the most promising transposon tools ever identified because piggyBac has the advantage that it can transpose without leaving a footprint at the excised site. Applying the piggyBac transposon to precise genome editing in plants, we have demonstrated efficient and precise piggyBac transposon excision from a transgene locus integrated into the rice genome. Furthermore, introduction of only desired point mutations into the target gene can be achieved by a combination of precise gene modification via homologous recombination-mediated gene targeting with subsequent marker excision from target loci using piggyBac transposition in rice. In addition, we have designed a piggyBac-mediated transgenesis system for the temporary expression of sequence-specific nucleases to eliminate the transgene from the host genome without leaving unnecessary sequences after the successful induction of targeted mutagenesis via sequence-specific nucleases for use in vegetatively propagated plants. In this review, we summarize our previous works and the future prospects of genetic engineering with piggyBac transposon.

Plant-Associated Bacillus thuringiensis and Bacillus cereus: Inside Agents for Biocontrol and Genetic Recombination in Phytomicrobiome

Bacillus thuringiensis Berliner (Bt) and B. cereus sensu stricto Frankland and Frankland are closely related species of aerobic, spore-forming bacteria included in the B. cereus sensu lato group. This group is one of the most studied, but it remains also the most mysterious species of bacteria. Despite more than a century of research on the features of these ubiquitous bacteria, there are a lot of questionable issues related to their taxonomy, resistance to external influences, endophytic existence, their place in multidimensional relationships in the ecosystem, and many others. The review summarizes current data on the mutualistic relationships of Bt and B. cereus bacteria with plants, the structure of the phytomicrobiomes including Bt and B. cereus, and the abilities of plant-associated and endophytic strains to improve plant resistance to various environmental factors and its productivity. Key findings on the possibility of the use of Cry gene promoter for transcription of the target dsRNA and simultaneous release of pore-forming proteins and provocation of RNA-interference in pest organisms allow us to consider this group of microorganisms as unique tools of genetic engineering and biological control. This will open the prospects for the development and direct change of plant microbiomes, and possibly serve as the basis for the regulation of the entire agroecosystem.

Metagenomics harvested genus-specific single-stranded DNA-annealing proteins improve and expand recombineering in Pseudomonas species.

The widespread Pseudomonas genus comprises a collection of related species with remarkable abilities to degrade plastics and polluted wastes and to produce a broad setof valuable compounds, ranging from bulk chemicals to pharmaceuticals. Pseudomonas possess characteristics of tolerance and stress resistance making them valuable hosts for industrial and environmental biotechnology. However, efficient and high-throughput genetic engineering tools have limited metabolic engineering efforts and applications. To improve their genome editing capabilities, we firstemployed a computational biology workflow to generate a genus-specific library of potential single-stranded DNA-annealing proteins (SSAPs). Assessment of the library was performed in different Pseudomonas using a high-throughput pooled recombinase screen followed by Oxford Nanopore NGS analysis. Among different active variants with variable levels of allelic replacement frequency (ARF), efficient SSAPs were found and characterized for mediating recombineering in the four tested species. New variants yielded higher ARFs than existing ones in Pseudomonas putida andPseudomonas aeruginosa, andexpanded the field of recombineering in Pseudomonas taiwanensisand Pseudomonas fluorescens. These findings will enhance the mutagenesis capabilities of these members of the Pseudomonas genus, increasing the possibilities for biotransformation and enhancingtheir potential for synthetic biology applications. .

Towards a new European Union’s regulation for certain NGT plant products

Ten years ago, biotechnologies experienced a technological breakthrough with the new genome-editing techniques (particularly CRISPR/Cas). This technological revolution not only opens up new fields of investigation with more powerful genetic engineering tools for human, animal and plant health, but also changes the regulatory criteria applied to genetic transformations and the legislation in many countries. Considering this new international background, the European Union was obliged to review Community regulations regarding these new genomic techniques (NGTs). The regulatory review process is long and complex. Begun in 2019, where are we in 2023? What genetic transformations are involved? Is the response up to the challenges? This article proposes some answers to these questions.

Securing maize reproductive success under drought stress by harnessing CO2 fertilization for greater productivity.

Securing maize grain yield is crucial to meet food and energy needs for the future growing population, especially under frequent drought events and elevated CO2 (eCO2) due to climate change. To maximize the kernel setting rate under drought stress is a key strategy in battling against the negative impacts. Firstly, we summarize the major limitations to leaf source and kernel sink in maize under drought stress, and identified that loss in grain yield is mainly attributed to reduced kernel set. Reproductive drought tolerance can be realized by collective contribution with a greater assimilate import into ear, more available sugars for ovary and silk use, and higher capacity to remobilize assimilate reserve. As such, utilization of CO2 fertilization by improved photosynthesis and greater reserve remobilization is a key strategy for coping with drought stress under climate change condition. We propose that optimizing planting methods and mining natural genetic variation still need to be done continuously, meanwhile, by virtue of advanced genetic engineering and plant phenomics tools, the breeding program of higher photosynthetic efficiency maize varieties adapted to eCO2 can be accelerated. Consequently, stabilizing maize production under drought stress can be achieved by securing reproductive success by harnessing CO2 fertilization.

Designing of future ornamental crops: a biotechnological driven perspective.

With a basis in human appreciation of beauty and aesthetic values, the new era of ornamental crops is based on implementing innovative technologies and transforming symbols into tangible assets. Recent advances in plant biotechnology have attracted considerable scientific and industrial interest, particularly in terms of modifying desired plant traits and developing future ornamental crops. By utilizing omics approaches, genomic data, genetic engineering, and gene editing tools, scientists have successively explored the underlying molecular mechanism and potential gene(s) behind trait regulation such as floral induction, plant architecture, stress resistance, plasticity, adaptation, and phytoremediation in ornamental crop species. These signs of progress lay a theoretical and practical foundation for designing and enhancing the efficiency of ornamental plants for a wide range of applications. In this review, we briefly summarized the existing literature and advances in biotechnological approaches for the improvement of vital traits in ornamental plants. The future ornamental plants, such as light-emitting plants, biotic/abiotic stress detectors, and pollution abatement, and the introduction of new ornamental varieties via domestication of wild species are also discussed.

CRISPR-Cas9 system: a novel and promising era of genotherapy for beta-hemoglobinopathies, hematological malignancy, and hemophilia.

Gene therapy represents a significant potential to revolutionize the field of hematology with applications in correcting genetic mutations, generating cell lines and animal models, and improving the feasibility and efficacy of cancer immunotherapy. Compared to different genetic engineering tools, clustered regularly interspaced short palindromic repeats (CRISPR) CRISPR-associated protein 9 (Cas9) emerged as an effective and versatile genetic editor with the ability to precisely modify the genome. The applications of genetic engineering in various hematological disorders have shown encouraging results. Monogenic hematological disorders can conceivably be corrected with single gene modification. Through the use of CRISPR-CAS9, restoration of functional red blood cells and hemostasis factors were successfully attained in sickle cell anemia, beta-thalassemia, and hemophilia disorders. Our understanding of hemato-oncology has been advanced via CRIPSR-CAS9 technology. CRISPR-CAS9 aided to build a platform of mutated genes responsible for cell survival and proliferation in leukemia. Therapeutic application of CRISPR-CAS9 when combined with chimeric antigen receptor (CAR) T cell therapy in multiple myeloma and acute lymphoblastic leukemia was feasible with attenuation of CAR T cell therapy pitfalls. Our review outlines the latest literature on the utilization of CRISPR-Cas9 in the treatment of beta-hemoglobinopathies and hemophilia disorders. We present the strategies that were employed and the findings of preclinical and clinical trials. Also, the review will discuss gene engineering in the field of hemato-oncology as a proper tool to facilitate and overcome the drawbacks of chimeric antigen receptor T cell therapy (CAR-T).

CRISPR-based tools for targeted genetic manipulation in pathogenic Sporothrix species.

Sporothrix brasiliensis is an emerging fungal pathogen frequently associated with zoonotic transmission of sporotrichosis by contaminated cats. Within 25 years, the disease has spread not only throughout Brazil but now to neighboring countries in Latin America. Thermo-dimorphism, melanin, glycans, adhesins, and secreted vesicles have been associated with the ability of Sporothrix species to cause disease in the mammalian host. Although certain virulence factors have been proposed as potential determinants for sporotrichosis, the scarcity of molecular tools for performing reverse genetics in Sporothrix has significantly impeded the dissection of mechanisms underlying the disease. Here, we demonstrate that PEG-mediated protoplast transformation is a powerful method for heterologous gene expression in S. brasiliensis, S. schenckii, and S. chilensis. Combined with CRISPR/Cas9 gene editing, this transformation protocol enabled the deletion of the putative DHN-melanin synthase gene pks1, which is a proposed virulence factor of Sporothrix species. To improve in locus integration of deletion constructs, we deleted the KU80 homolog that is critical for non-homologous end-joining DNA repair. The use of Δku80 strains from S. brasiliensis enhanced homologous-directed repair during transformation resulting in increased targeted gene deletion in combination with CRISPR/Cas9. In conclusion, our CRISPR/Cas9-based transformation protocol provides an efficient tool for targeted gene manipulation in Sporothrix species. IMPORTANCE Sporotrichosis caused by Sporothrix brasiliensis is a disease that requires long periods of treatment and is rapidly spreading across Latin America. The virulence of this fungus and the surge of atypical and more severe presentations of the disease raise the need for an understanding of the molecular mechanisms underlying sporotrichosis, as well as the development of better diagnostics and antifungal therapies. By developing molecular tools for accurate genetic manipulation in Sporothrix, this study addresses the paucity of reliable and reproducible tools for stable genetic engineering of Sporothrix species, which has represented a major obstacle for studying the virulence determinants and their roles in the establishment of sporotrichosis.

Harnessing the Native Type I-F CRISPR-Cas System of Acinetobacter baumannii for Genome Editing and Gene Repression

The rapid emergence of infections caused by MDR Acinetobacter baumannii poses a serious threat to global public health. Therefore, it has become important to obtain a deeper understanding of the mechanisms of multidrug resistance and pathogenesis of A. baumannii. However, there are still relatively few genetic engineering tools for A. baumannii. Although A. baumannii possesses Type I-F CRISPR-Cas systems, they have not yet been used for genetic modifications. Here, we developed a single plasmid-mediated native Type I-F CRISPR-Cas system for gene editing and gene regulation in A. baumannii. The protospacer adjacent motif (PAM) sequence was identified as 5'-NCC-3' by analysis of the CRISPR array. Through introduction of the RecAb homologous recombination system, we significantly increased the knockout efficiency of the oxyR gene from 12.5% to 75.0% in A. baumannii. To investigate transcriptional inhibition by the Type I-F CRISPR system, we deleted the gene encoding its Cas2-3 nuclease and repurposed the native Type I-F Cascade effector to regulate transcription of alcohol dehydrogenase gene adh4. The level of adh4 transcription was inhibited by up to 900-fold compared to the control. The Cascade transcriptional module was also successfully applied in a clinical Klebsiella pneumoniae isolate. This study proposed a tool for future exploration of the genetic characteristics of A. baumannii or other clinical strains.

Functional characterization of the MSP-C6 promoter as a potential tool for mesocarp-preferential expression of transgenes

Modification of lipid composition in the mesocarp tissue of oil palm involves genetic manipulation of multiple genes. More than one mesocarp-preferential promoter is necessary for the expression of individual transgenes in the same plant to obviate transcriptional gene silencing. This study aimed to identify genes that are preferentially expressed in the mesocarp tissue and characterize selected candidate mesocarp-preferential promoters. Ten transcripts that were preferentially expressed in the mesocarp tissue were identified from the analysis of 82 transcriptome datasets of 12 different oil palm tissues. The expression of two candidate genes, MSP-C1 and MSP-C6, was verified to be preferentially expressed in the mesocarp tissues and shown to have a low expression level in non-mesocarp tissues by reverse transcription quantitative real-time PCR (RT-qPCR). MSP-C6 promoter fragments of different lengths were transformed into tomato plants for further characterization. Both unripe and ripe fruits of transgenic tomato plants transformed with a construct harboring the MSP-C6-F1 (2014 bp) promoter were shown to have high beta-glucuronidase (GUS) activities. The findings of this study suggest the potential applications of the MSP-C6 promoter as a molecular tool for genetic engineering of novel traits in fruit crops.

Engineered Bacteria: General Overview as Therapeutic Agent and a Novel Drug Delivery System.

Bacterial engineering modifies bacteria's genomic sequence using genetic engineering tools. These engineered bacteria can produce modified proteins, peptides, nucleic acids, and other biomolecules that can be used to treat various medical conditions. Engineered bacteria can target diseased tissues or organs, detect specific biomarkers in the diseased environment, and even induce specific conditions. Furthermore, a meticulously designed intracellular metabolic pathway can activate or inhibit the expression of related genes, synthesise biologically active therapeutic molecules, and precisely deliver drug payloads to diseased tissues or organs. Lactococcus (L. lactis), Salmonella (S. typhi)</i>, and E. coli (E. coli Nissle)</i> are the most studied engineered microorganisms used as drug carriers. These have been used in vaccines to treat multifactorial diseases such as cancer, autoimmune diseases, metabolic diseases, and inflammatory conditions. Other promising strains include Bifidobacterium animalis</i>, Listeria monocytogenes, Staphylococcus epidermidis, Staphylococcus lugdunensis</i>, and Clostridium sporogenes</i>. Despite the low reported risk, toxic effects associated with bacterial cells, limiting their efficacy and rapid clearance due to immune responses stimulated by high bacterial concentrations, remain major drawbacks. As a result, a better and more effective method of drug delivery must be developed by combining bacterial-based therapies with other available treatments, and more research in this area is also needed.

Construction of a bacteriophage-derived recombinase system in Bacillus licheniformis for gene deletion

Bacillus licheniformis and its related strains have found extensive applications in diverse industries, agriculture, and medicine. However, the current breeding methods for this strain primarily rely on natural screening and traditional mutagenesis. The limited availability of efficient genetic engineering tools, particularly recombination techniques, has hindered further advancements in its applications. In this study, we conducted a comprehensive investigation to identify and characterize a recombinase, RecT, derived from a Bacillus phage. Remarkably, the recombinase exhibited a 105-fold enhancement in the recombination efficiency of the strain. To facilitate genome editing, we developed a system based on the conditional expression of RecT using a rhamnose-inducible promoter (Prha). The efficacy of this system was evaluated by deleting the amyL gene, which encodes an α-amylase. Our findings revealed that the induction time and concentration of rhamnose, along with the generation time of the strain, significantly influenced the editing efficiency. Optimal conditions for genome editing were determined as follows: the wild-type strain was initially transformed with the genome editing plasmid, followed by cultivation and induction with 1.5% rhamnose for 8 h. Subsequently, the strain was further cultured for an additional 24 h, equivalent to approximately three generations. Consequently, the recombination efficiency reached an impressive 16.67%. This study represents a significant advancement in enhancing the recombination efficiency of B. licheniformis through the utilization of a RecT-based recombination system. Moreover, it provides a highly effective genome editing tool for genetic engineering applications in this strain.

From Nature to Lab: Sustainable Bacterial Cellulose Production and Modification with Synthetic Biology.

Bacterial cellulose (BC) is a macromolecule with versatile applications in medicine, pharmacy, biotechnology, cosmetology, food and food packaging, ecology, and electronics. Although many bacteria synthesize BC, the most efficient BC producers are certain species of the genera Komagataeibacter and Novacetimonas. These are also food-grade bacteria, simplifying their utilization at industrial facilities. The basic principles of BC synthesis are known from studies of Komagataeibacter xylinus, which became a model species for studying BC at genetic and molecular levels. Cellulose can also be of plant origin, but BC surpasses its purity. Moreover, the laboratory production of BC enables in situ modification into functionalized material with incorporated molecules during its synthesis. The possibility of growing Komagataeibacter and Novacetimonas species on various organic substrates and agricultural and food waste compounds also follows the green and sustainable economy principles. Further intervention into BC synthesis was enabled by genetic engineering tools, subsequently directing it into the field of synthetic biology. This review paper presents the development of the fascinating field of BC synthesis at the molecular level, seeking sustainable ways for its production and its applications towards genetic modifications of bacterial strains for producing novel types of living biomaterials using the flexible metabolic machinery of bacteria.

Uncovering transcriptional reprogramming during callus development in soybean: insights and implications.

Callus, a valuable tool in plant genetic engineering, originates from dedifferentiated cells. While transcriptional reprogramming during callus formation has been extensively studied in Arabidopsis thaliana, our knowledge of this process in other species, such as Glycine max, remains limited. To bridge this gap, our study focused on conducting a time-series transcriptome analysis of soybean callus cultured for various durations (0, 1, 7, 14, 28, and 42 days) on a callus induction medium following wounding with the attempt of identifying genes that play key roles during callus formation. As the result, we detected a total of 27,639 alterations in gene expression during callus formation, which could be categorized into eight distinct clusters. Gene ontology analysis revealed that genes associated with hormones, cell wall modification, and cell cycle underwent transcriptional reprogramming throughout callus formation. Furthermore, by scrutinizing the expression patterns of genes related to hormones, cell cycle, cell wall, and transcription factors, we discovered that auxin, cytokinin, and brassinosteroid signaling pathways activate genes involved in both root and shoot meristem development during callus formation. In summary, our transcriptome analysis provides significant insights into the molecular mechanisms governing callus formation in soybean. The information obtained from this study contributes to a deeper understanding of this intricate process and paves the way for further investigation in the field.

Eras of designer Tregs: Harnessing synthetic biology for immune suppression.

Since their discovery, CD4+ CD25hi FOXP3hi regulatory T cells (Tregs) have been firmly established as a critical cell type for regulating immune homeostasis through a plethora of mechanisms. Due to their immunoregulatory power, delivery of polyclonal Tregs has been explored as a therapy to dampen inflammation in the settings of transplantation and autoimmunity. Evidence shows that Treg therapy is safe and well-tolerated, but efficacy remains undefined and could be limited by poor persistence in vivo and lack of antigen specificity. With the advent of new genetic engineering tools, it is now possible to create bespoke "designer" Tregs that not only overcome possible limitations of polyclonal Tregs but also introduce new features. Here, we review the development of designer Tregs through the perspective of three 'eras': (1) the era of FOXP3 engineering, in which breakthroughs in the biological understanding of this transcription factor enabled the conversion of conventional T cells to Tregs; (2) the antigen-specificity era, in which transgenic T-cell receptors and chimeric antigen receptors were introduced to create more potent and directed Treg therapies; and (3) the current era, which is harnessing advanced genome-editing techniques to introduce and refine existing and new engineering approaches. The year 2022 marked the entry of "designer" Tregs into the clinic, with exciting potential for application and efficacy in a wide variety of immune-mediated diseases.

Host-Delivered RNA Interference for Durable Pest Resistance in Plants: Advanced Methods, Challenges, and Applications.

Insect-pests infestation greatly affects global agricultural production and is projected to become more severe in upcoming years. There is concern about pesticide application being ineffective due to insect resistance and environmental toxicity. Reduced effectiveness of Bt toxins also made the scientific community shift toward alternative strategies to control devastating agricultural pests. With the advent of host-delivered RNA interference, also known as host-induced gene silencing, targeted insect genes have been suppressed through genetic engineering tools to deliver a novel insect-pest resistance strategy for combating a number of agricultural pests. This review recapitulates the possible mechanism of host-delivered RNA interference (HD-RNAi), in particular, the silencing of target genes of insect-pests. We emphasize the development of the latest strategies against evolving insect targets including designing of artificial microRNAs, vector constructs, and the benefit of using plastid transformation to transform target RNA-interfering genes. Advantages of using HD-RNAi over other small RNA delivery modes and also the supremacy of HD-RNAi over the CRISPR-Cas system particularly for insect resistance have been described. However, the broader application of this technology is restricted due to its several limitations. Using artificial miRNA designs, the host-delivered RNAi + Bt combinatorial approach and chloroplast transformation can overcome limitations of RNAi. With careful design and delivery approaches, RNAi promises to be extremely valuable and effectiveplant protection strategy toattain durable insect-pest resistance in crops.