A Computational Approach for Designing Synthetic Riboswitches for Next-Generation RNA Therapeutics.

Riboswitches are naturally occurring regulatory segments of RNA molecules that modulate gene expression in response to specific ligand binding. They serve as a molecular 'switch' that controls the RNA's structure and function, typically influencing the synthesis of proteins. Riboswitches are unique because they directly interact with metabolites without the need for proteins, making them attractive tools in synthetic biology and RNA-based therapeutics. In synthetic biology, riboswitches are harnessed to create biosensors and genetic circuits. Their ability to respond to specific molecular signals allows for the design of precise control mechanisms in genetic engineering. This specificity is particularly useful in therapeutic applications, where riboswitches can be synthetically designed to respond to disease-specific metabolites, thereby enabling targeted drug delivery or gene therapy. Advancements in designing synthetic riboswitches for RNA-based therapeutics hinge on sophisticated computational techniques, which are described in this chapter. The chapter concludes by underscoring the potential of computational strategies in revolutionizing the design and application of synthetic riboswitches, paving the way for advanced RNA-based therapeutic solutions.

Genetically engineered CD276-anchoring biomimetic nanovesicles target senescent escaped tumor cells to overcome chemoresistant and immunosuppressive breast cancer.

Chemotherapy-induced cellular senescence leads to an increased proportion of cancer stem cells (CSCs) in breast cancer (BC), contributing to recurrence and metastasis, while effective means to clear them are currently lacking. Herein, we aim to develop new approaches for selectively killing senescent-escape CSCs. High CD276 (95.60%) expression in multidrug-resistant BC cells, facilitates immune evasion by low-immunogenic senescent escape CSCs. CALD1, upregulated in ADR-resistant BC, promoting senescent-escape of CSCs with an anti-apoptosis state and upregulating CD276, PD-L1 to promote chemoresistance and immune escape. We have developed a controlled-released thermosensitive hydrogel containing pH- responsive anti-CD276 scFV engineered biomimetic nanovesicles to overcome BC in primary, recurrent, metastatic and abscopal humanized mice models. Nanovesicles coated anti-CD276 scFV selectively fuses with cell membrane of senescent-escape CSCs, then sequentially delivers siCALD1 and ADR due to pH-responsive MnP shell. siCALD1 together with ADR effectively induce apoptosis of CSCs, decrease expression of CD276 and PD-L1, and upregulate MHC I combined with Mn2+ to overcome chemoresistance and promote CD8+T cells infiltration. This combined therapeutic approach reveals insights into immune surveillance evasion by senescent-escape CSCs, offering a promising strategy to immunotherapy effectiveness in cancer therapy.

Orthogonal Serine Integrases Enable Scalable Gene Storage Cascades in Bacterial Genome.

Genome integration enables host organisms to stably carry heterologous DNA messages, introducing new genotypes and phenotypes for expanded applications. While several genome integration approaches have been reported, a scalable tool for DNA message storage within site-specific genome landing pads is still lacking. Here, we introduce an iterative genome integration method utilizing orthogonal serine integrases, enabling the stable storage of multiple heterologous genes in the chromosome of Escherichia coli MG1655. By leveraging serine integrases TP901-1, Bxb1, and PhiC31, along with engineered integration vectors, we demonstrate high-efficiency, marker-free integration of DNA fragments up to 13 kb in length. To further simplify the procedure, we then develop a streamlined integration method and showcase the system's versatility by constructing an engineered E. coli strain capable of storing and expressing multiple genes from diverse species. Additionally, we illustrate the potential utility of these engineered strains for synthetic biology applications, including in vivo and in vitro protein expression. Our work extends the application scope of serine integrases for scalable gene integration cascades, with implications for genome manipulation and gene storage applications in synthetic biology.

Challenges to rhizobial adaptability in a changing climate: Genetic engineering solutions for stress tolerance.

Rhizobia interact with leguminous plants in the soil to form nitrogen fixing nodules in which rhizobia and plant cells coexist. Although there are emerging studies on rhizobium-associated nitrogen fixation in cereals, the legume-rhizobium interaction is more well-studied and usually serves as the model to study rhizobium-mediated nitrogen fixation in plants. Rhizobia play a crucial role in the nitrogen cycle in many ecosystems. However, rhizobia are highly sensitive to variations in soil conditions and physicochemical properties (i.e. moisture, temperature, salinity, pH, and oxygen availability). Such variations directly caused by global climate change are challenging the adaptive capabilities of rhizobia in both natural and agricultural environments. Although a few studies have identified rhizobial genes that confer adaptation to different environmental conditions, the genetic basis of rhizobial stress tolerance remains poorly understood. In this review, we highlight the importance of improving the survival of rhizobia in soil to enhance their symbiosis with plants, which can increase crop yields and facilitate the establishment of sustainable agricultural systems. To achieve this goal, we summarize the key challenges imposed by global climate change on rhizobium-plant symbiosis and collate current knowledge of stress tolerance-related genes and pathways in rhizobia. And finally, we present the latest genetic engineering approaches, such as synthetic biology, implemented to improve the adaptability of rhizobia to changing environmental conditions.

[Biosafety risks, control, and biocontainment of engineered microalgae].

Microalgae, with the ability to harness solar energy to fix CO2 and convert it into organic compounds, have emerged as promising green cell factories. With the rapid development of cutting-edge biotechnologies, the research and application of photosynthetic microalgae have been expanding, leading to comprehensive and in-depth engineering of microalgae. The synthetic biology and genome editing technologies have enabled the applications of microalgae in medicine, agriculture, food, energy, and the environment. However, the survival and spreading of engineered microalgae in the natural environment pose potential safety risks to ecosystems and human health. To curb the risks caused by the spreading of engineered microalgae in the environment, biosafety policies should be formulated for engineered microalgae and the prevention and control technologies should be developed. Toward this goal, researchers have developed biocontainment systems, including positive strategies such as the design of toxic protein-based kill switches and passive strategies such as knocking out essential genes to construct the strains with nutritional deficiencies, thereby spatially containing engineered microalgae. This article summarizes the application of cutting-edge biotechnologies in the engineering of microalgae, the biosafety risks and management regulations associated with the escape of engineered microalgae, and the progress in novel biocontainment technologies established for engineered microalgae. Finally, this article gives insights into the future development direction of microalgae biocontainment.

Integrating Deep Learning and Synthetic Biology: A Co-Design Approach for Enhancing Gene Expression via N-Terminal Coding Sequences.

N-terminal coding sequence (NCS) influences gene expression by impacting the translation initiation rate. The NCS optimization problem is to find an NCS that maximizes gene expression. The problem is important in genetic engineering. However, current methods for NCS optimization such as rational design and statistics-guided approaches are labor-intensive yield only relatively small improvements. This paper introduces a deep learning/synthetic biology codesigned few-shot training workflow for NCS optimization. Our method utilizes k-nearest encoding followed by word2vec to encode the NCS, then performs feature extraction using attention mechanisms, before constructing a time-series network for predicting gene expression intensity, and finally a direct search algorithm identifies the optimal NCS with limited training data. We took green fluorescent protein (GFP) expressed by Bacillus subtilis as a reporting protein of NCSs, and employed the fluorescence enhancement factor as the metric of NCS optimization. Within just six iterative experiments, our model generated an NCS (MLD62) that increased average GFP expression by 5.41-fold, outperforming the state-of-the-art NCS designs. Extending our findings beyond GFP, we showed that our engineered NCS (MLD62) can effectively boost the production of N-acetylneuraminic acid by enhancing the expression of the crucial rate-limiting GNA1 gene, demonstrating its practical utility. We have open-sourced our NCS expression database and experimental procedures for public use.

Concurrent Oncolysis and Neurolesion Repair by Dual Gene-Engineered hNSCs in an Experimental Model of Intraspinal Cord Glioblastoma.

Intramedullary spinal cord glioblastoma (ISCG) is lethal due to lack of effective treatment. We previously established a rat C6-ISCG model and the antitumor effect of F3.CD-TK, an hNSC line expressing CD and TK, via producing cytocidal 5FU and GCV-TP. However, the neurotherapeutic potential of this hNSC approach has remained uninvestigated. Here for the first time, cultured F3.CD-TK cells were found to have a markedly higher oncolytic effect, which was GJIC-dependent, and BDNF expression but less VEGF secretion than F3.CD. In Rowett athymic rats, F3.CD-TK (1.5 × 106 cells/10 µL × 2), injected near C6-ISCG (G55 seeding 7 days earlier: 10 K/each) and followed by q.d. (×5/each repeat; i.p.) of 5FC (500 mg/kg/5 mL/day) and GCV (25 mg/kg/1 mL/day), robustly mitigated cardiorespiratory, locomotor, and sensory deficits to improve neurofunction and overall survival compared to animals receiving either F3.CD or F3.CD-TK+F3.CD debris formula. The F3.CD-TK regimen exerted greater tumor penetration and neural inflammation/immune modulation, reshaped C6-ISCG topology to increase the tumor's surface area/volume ratio to spare/repair host axons (e.g., vGlut1+ neurites), and had higher post-prodrug donor self-clearance. The multimodal data and mechanistic leads from this proof-of-principle study suggest that the overall stronger anti-ISCG benefit of our hNSC-based GDEPT is derived from its concurrent oncolytic and neurotherapeutic effects.

Monitoring Electroporation-Induced Changes in Action Potential Generation in Genetically Engineered Tet-On Spiking HEK cells.

Excitable cells such as neuronal and muscle cells can be primary targets in rapidly emerging electroporation-based treatments. However, they can be affected by electric pulses even in therapies where they are not the primary targets, and this can cause adverse side effects. Therefore, to optimize the electroporation-based treatments of excitable and non-excitable tissues, there is a need to study the effects of electric pulses on excitable cells, their ion channels, and excitability in vitro. For this purpose, a protocol was developed for optical monitoring of changes in action potential generation due to electroporation on a simple excitable cell model of genetically engineered tet-on spiking HEK cells. With the use of a fluorescent potentiometric dye, the changes in transmembrane voltage were monitored under a fluorescence microscope, and relevant parameters of cell responses were extracted automatically with a MATLAB application. This way, the excitable cell responses to different electric pulses and the interplay between excitation and electroporation could be efficiently evaluated.

An improved CRISPR and CRISPR interference (CRISPRi) toolkit for engineering the model methanogenic archaeon Methanococcus maripaludis.

BACKGROUND: The type II based CRISPR-Cas system remains restrictedly utilized in archaea, a featured domain of life that ranks parallelly with Bacteria and Eukaryotes. Methanococcus maripaludis, known for rapid growth and genetic tractability, serves as an exemplary model for studying archaeal biology and exploring CO2-based biotechnological applications. However, tools for controlled gene regulation remain deficient and CRISPR-Cas tools still need improved in this archaeon, limiting its application as an archaeal model cellular factory. RESULTS: This study not only improved the CRISPR-Cas9 system for optimizing multiplex genome editing and CRISPR plasmid construction efficiencies but also pioneered an effective CRISPR interference (CRISPRi) system for controlled gene regulation in M. maripaludis. We developed two novel strategies for balanced expression of multiple sgRNAs, facilitating efficient multiplex genome editing. We also engineered a strain expressing Cas9 genomically, which simplified the CRISPR plasmid construction and facilitated more efficient genome modifications, including markerless and scarless gene knock-in. Importantly, we established a CRISPRi system using catalytic inactive dCas9, achieving up to 100-fold repression on target gene. Here, sgRNAs targeting near and downstream regions of the transcription start site and the 5'end ORF achieved the highest repression efficacy. Furthermore, we developed an inducible CRISPRi-dCas9 system based on TetR/tetO platform. This facilitated the inducible gene repression, especially for essential genes. CONCLUSIONS: Therefore, these advancements not only expand the toolkit for genetic manipulation but also bridge methodological gaps for controlled gene regulation, especially for essential genes, in M. maripaludis. The robust toolkit developed here paves the way for applying M. maripaludis as a vital model archaeal cell factory, facilitating fundamental biological studies and applied biotechnology development of archaea.

Advances in CRISPR-Cas systems for fungal infections.

Fungi contain a wide range of bioactive secondary metabolites (SMs) that have numerous applications in various fields, including agriculture, medicine, human health, and more. It is common for genes responsible for the production of secondary metabolites (SMs) to form biosynthetic gene clusters (BGCs). The identification and analysis of numerous unexplored gene clusters (BGCs) and their corresponding substances (SMs) has been significantly facilitated by the recent advancements in genomic and genetic technologies. Nevertheless, the exploration of secondary metabolites with commercial value is impeded by a variety of challenges. The emergence of modern CRISPR/Cas technologies has brought about a paradigm shift in fungal genetic engineering, significantly streamlining the process of discovering new bioactive compounds. This study begins with an examination of fungal biosynthetic gene clusters (BGCs) and their interconnections with the secondary metabolites (SMs) they generate. Following that, a brief summary of the conventional methods employed in fungal genetic engineering is provided. This study explores various sophisticated CRISPR/Cas-based methodologies and their utilization in examining the synthesis of secondary metabolites (SMs) in fungi. The chapter provides an in-depth analysis of the limitations and obstacles encountered in CRISPR/Cas-based systems when applied to fungal genetic engineering. It also proposes promising avenues for future research to optimize the efficiency of these systems.

Engineering Rhodosporidium toruloides for sustainable production of value-added punicic acid from glucose and wood residues.

Rhodosporidium toruloides has emerged as a prominent candidate for producing single-cell oil from cost-effective feedstocks. In this study, the capability of R. toruloides to produce punicic acid (PuA), a representative plant unusual fatty acid, was investigated. The introduction of acyl lipid desaturase and conjugase (PgFADX) allowed R. toruloides to accumulate 3.7 % of total fatty acids as PuA. Delta-12 acyl lipid desaturase (PgFAD2) and diacylglycerol acyltransferase 2 were shown to benefit PuA production. The strain with PgFADX and PgFAD2 coexpression accumulated 12 % of its lipids as PuA from glucose, which translated into a PuA titer of 451.6 mg/L in shake flask condition. Utilizing wood hydrolysate as the feedstock, this strain produced 6.4 % PuA with a titer of 310 mg/L. Taken together, the results demonstrated that R. toruloides could serve as an ideal platform for the production of plant-derived high-value conjugated fatty acid using agricultural and forestry waste as feedstock.

Temporal epigenome modulation enables efficient bacteriophage engineering and functional analysis of phage DNA modifications.

Lytic bacteriophages hold substantial promise in medical and biotechnological applications. Therefore a comprehensive understanding of phage infection mechanisms is crucial. CRISPR-Cas systems offer a way to explore these mechanisms via site-specific phage mutagenesis. However, phages can resist Cas-mediated cleavage through extensive DNA modifications like cytosine glycosylation, hindering mutagenesis efficiency. Our study utilizes the eukaryotic enzyme NgTET to temporarily reduce phage DNA modifications, facilitating Cas nuclease cleavage and enhancing mutagenesis efficiency. This approach enables precise DNA targeting and seamless point mutation integration, exemplified by deactivating specific ADP-ribosyltransferases crucial for phage infection. Furthermore, by temporally removing DNA modifications, we elucidated the effects of these modifications on T4 phage infections without necessitating gene deletions. Our results present a strategy enabling the investigation of phage epigenome functions and streamlining the engineering of phages with cytosine DNA modifications. The described temporal modulation of the phage epigenome is valuable for synthetic biology and fundamental research to comprehend phage infection mechanisms through the generation of mutants.

Systematic multi-trait AAV capsid engineering for efficient gene delivery.

Broadening gene therapy applications requires manufacturable vectors that efficiently transduce target cells in humans and preclinical models. Conventional selections of adeno-associated virus (AAV) capsid libraries are inefficient at searching the vast sequence space for the small fraction of vectors possessing multiple traits essential for clinical translation. Here, we present Fit4Function, a generalizable machine learning (ML) approach for systematically engineering multi-trait AAV capsids. By leveraging a capsid library that uniformly samples the manufacturable sequence space, reproducible screening data are generated to train accurate sequence-to-function models. Combining six models, we designed a multi-trait (liver-targeted, manufacturable) capsid library and validated 88% of library variants on all six predetermined criteria. Furthermore, the models, trained only on mouse in vivo and human in vitro Fit4Function data, accurately predicted AAV capsid variant biodistribution in macaque. Top candidates exhibited production yields comparable to AAV9, efficient murine liver transduction, up to 1000-fold greater human hepatocyte transduction, and increased enrichment relative to AAV9 in a screen for liver transduction in macaques. The Fit4Function strategy ultimately makes it possible to predict cross-species traits of peptide-modified AAV capsids and is a critical step toward assembling an ML atlas that predicts AAV capsid performance across dozens of traits.

One-step genome engineering in bee gut bacterial symbionts.

Mechanistic understanding of interactions in many host-microbe systems, including the honey bee microbiome, is limited by a lack of easy-to-use genome engineering approaches. To this end, we demonstrate a one-step genome engineering approach for making gene deletions and insertions in the chromosomes of honey bee gut bacterial symbionts. Electroporation of linear or non-replicating plasmid DNA containing an antibiotic resistance cassette flanked by regions with homology to a symbiont genome reliably results in chromosomal integration. This lightweight approach does not require expressing any exogenous recombination machinery. The high concentrations of large DNAs with long homology regions needed to make the process efficient can be readily produced using modern DNA synthesis and assembly methods. We use this approach to knock out genes, including genes involved in biofilm formation, and insert fluorescent protein genes into the chromosome of the betaproteobacterial bee gut symbiont Snodgrassella alvi. We are also able to engineer the genomes of multiple strains of S. alvi and another species, Snodgrassella communis, which is found in the bumble bee gut microbiome. Finally, we use the same method to engineer the chromosome of another bee symbiont, Bartonella apis, which is an alphaproteobacterium. As expected, gene knockout in S. alvi using this approach is recA-dependent, suggesting that this straightforward procedure can be applied to other microbes that lack convenient genome engineering methods. IMPORTANCE: Honey bees are ecologically and economically important crop pollinators with bacterial gut symbionts that influence their health. Microbiome-based strategies for studying or improving bee health have utilized wild-type or plasmid-engineered bacteria. We demonstrate that a straightforward, single-step method can be used to insert cassettes and replace genes in the chromosomes of multiple bee gut bacteria. This method can be used for investigating the mechanisms of host-microbe interactions in the bee gut community and stably engineering symbionts that benefit pollinator health.

Molecular protocol to develop ß-carotene-biofortified rice events via molecular optimization.

Providing food with nutrition and functionality is crucial for sustaining human life. Rice (Oryza sativa L.) is a representative staple crop with high carbohydrate content but low amounts of essential amino acids, micronutrients, and carotenoids such as provitamin A. To improve the nutritional quality, rice endosperm was biofortified to accumulate carotenoids such as ß-carotene through genetic engineering (i.e., using synthetic carotenoid biosynthetic genes, a nonmammalian viral polycistronic sequence, and an optimized promoter and transit peptide) and high-throughput rice transformation (approximately 300 transgenic plants per construct). To facilitate the safety assessment of genetically modified food, molecular characterization was performed to select elite lines equipped with a single intergenic insertion of T-DNA, high transgene expression, in this case leading to high carotenoid content, and with phenotypic and compositional substantial equivalence. In this study, we present ß-carotene-biofortified rice event candidate lines eligible for commercial use and a disclosed molecular protocol for the development of biotech rice crops.

Biotechnological approaches for enhancement of heavy metal phytoremediation capacity of plants.

Heavy metals are extremely hazardous for human health due to their toxic effects. They are non-biodegradable in nature, thus remain in the environment and enter and accumulate in the human body through biomagnification; hence, there is a serious need of their remediation. Phytoremediation has emerged as a green, sustainable, and effective solution for heavy metal removal and many plant species could be employed for this purpose. Plants are able to sequester substantial quantity of heavy metals, in some cases thousands of ppm, due to their robust physiology enabling high metal tolerance and anatomy supporting metal ion accumulation. Identification and modification of potential target genes involved in heavy metal accumulation have led to improved phytoremediation capacity of plants at the molecular level. The introduction of foreign genes through genetic engineering approaches has further enhanced phytoremediation capacity manifolds. This review gives an insight towards improving the phytoremediation efficiency through a better understanding of molecular mechanisms involved, expression of different proteins, genetic engineering approaches for transgenic production, and genetic modifications. It also comprehends novel omics tools such as genomics, metabolomics, proteomics, transcriptomics, and genome editing technologies for improvement of phytoremediation ability of plants.

Cracking the Code: Reprogramming the Genetic Script in Prokaryotes and Eukaryotes to Harness the Power of Noncanonical Amino Acids.

Over 500 natural and synthetic amino acids have been genetically encoded in the last two decades. Incorporating these noncanonical amino acids into proteins enables many powerful applications, ranging from basic research to biotechnology, materials science, and medicine. However, major challenges remain to unleash the full potential of genetic code expansion across disciplines. Here, we provide an overview of diverse genetic code expansion methodologies and systems and their final applications in prokaryotes and eukaryotes, represented by Escherichia coli and mammalian cells as the main workhorse model systems. We highlight the power of how new technologies can be first established in simple and then transferred to more complex systems. For example, whole-genome engineering provides an excellent platform in bacteria for enabling transcript-specific genetic code expansion without off-targets in the transcriptome. In contrast, the complexity of a eukaryotic cell poses challenges that require entirely new approaches, such as striving toward establishing novel base pairs or generating orthogonally translating organelles within living cells. We connect the milestones in expanding the genetic code of living cells for encoding novel chemical functionalities to the most recent scientific discoveries, from optimizing the physicochemical properties of noncanonical amino acids to the technological advancements for their in vivo incorporation. This journey offers a glimpse into the promising developments in the years to come.

A Synthetic Biology Approach to Transgene Expression in Insects.

The ability to control gene expression is pivotal in genetic engineering and synthetic biology. However, in most nonmodel 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 5' regulatory sequences, representing a significant advancement in mosquito synthetic biology gene expression tools.

Protocol for genetic engineering in Drosophila suzukii using microinjection.

The spotted wing Drosophila (Drosophila suzukii Matsumura) is recognized globally as a significant economic pest. Here, we present a protocol for genetic engineering in D. suzukii using microinjection. We describe steps for genetic engineering techniques, including transposon-mediated germline transformation, recombinase-mediated genome targeting, and CRISPR-mediated gene editing. This protocol can significantly expand the toolkit for functional genomics and genetic control studies of this pest. For complete details on the use and execution of this protocol, please refer to Schetelig and Handler,1 Schetelig et al.2 Yan et al.,3 and Yan et al.4.

Synthetic Biology of Natural Products Engineering: Recent Advances Across the Discover-Design-Build-Test-Learn Cycle.

Advances in genome engineering and associated technologies have reinvigorated natural products research. Here we highlight the latest developments in the field across the discover-design-build-test-learn cycle of bioengineering, from recent progress in computational tools for AI-supported genome mining, enzyme and pathway engineering, and compound identification to novel host systems and new techniques for improving production levels, and place these trends in the context of responsible research and innovation, emphasizing the importance of anticipatory analysis at the early stages of process development.