The environmental and therapeutic application of genetically engineered microorganisms necessitates the development of robust, irreversible biocontainment systems. In this study, we present an eEGM (editing-driven essential gene multiplex inactivation) module that utilizes CRISPR-mediated cytidine base editing to induce permanent self-killing via a single transient induction. By targeting the start codons of essential genes, we achieved an irreversible translational blockade that avoids the fitness costs associated with basal toxicity in nuclease-based systems. Multiplexed targeting of non-redundant essential loci (holA, ftsB, and dfp) yielded escape frequencies at or below the NIH guideline criterion (10−8) within 1 h of pulse induction. Furthermore, the eEGM system exhibited robust functional orthogonality and portability across laboratory, industrial, and therapeutic E. coli strains, including MG1655, W3110, and Nissle 1917, without detectable interference with heterologous protein expression. This work establishes base editing as a cleavage-free CRISPR effector for pulse-activated, irreversible biocontainment and provides a practical framework for safer deployment of engineered microbes.

This study presents an integrated waste-to-energy strategy for the sustainable conversion of the biodegradable organic fraction of municipal solid waste (BOFMSW) into biohydrogen (Bio.H2) and biomethane (Bio.CH4) through a two-stage continuous stirred dark fermentation (DF) process. The first-stage bioreactor was inoculated with Clostridium Thermocellum selectively enriched in a 2-bromoethanesulfonic acid (BESA) medium, and the influence of bimetallic ion catalysts NiCl2 + FeCl2 and NiCl2 + FeSO4 was evaluated at various concentrations (25, 50, 75, and 100 mg/L). The catalyst combination NiCl2 + FeCl2 at 75 mg/L produced the maximum Bio.H2 yield of 3162 L, representing a 69% enhancement compared with the catalyst-free substrate. At this optimal catalytic concentration, the percentage of H2 in the gas composition was 69.26%. The second-stage bioreactor utilized the effluent from the first stage for Bio.CH4 generation, achieving the highest cumulative yield of 729 L at 50 mg/L NiCl2 + FeCl2, which was 59% higher than that of the catalyst-free substrate. The two-stage process achieved an overall COD removal efficiency of 93.18%, demonstrating the system’s effective capacity for energy recovery. Fourier Transform Infrared (FTIR) and Field Emission Scanning Electron Microscopy (FESEM) analyses confirmed the biochemical and morphological degradation of complex organics into volatile fatty acids, illustrating efficient substrate conversion and microbial proliferation. The final digested slurry, rich in nitrogen, phosphorus, and potassium, was found suitable for use as a bio-fertilizer, supporting nutrient recycling and soil enrichment. This integrated process not only improved energy recovery efficiency but also achieved near-zero waste discharge, combining waste-to-energy and waste-to-resource approaches. The developed system demonstrates strong potential for pilot-scale implementation of Bio.H2, and Bio.CH4 for co-production from municipal solid waste (MSW), offering a circular bioeconomy pathway toward low-carbon, sustainable urban waste management.

Extracellular vesicles (EVs) are cell-released lipid-wrapped nano- to micro-sized particles without self-replication ability. Plant cells secrete EVs (plant EVs, PEVs) during normal development of plants and in plant response to external stimuli. Distinct from plant-derived nanoparticles, here we narrow the definition of PEVs, which are naturally secreted by cells, excluding those from disrupted cells or artificial vesicles with properties similar to those of EVs. We focus on PEV classification, isolation methods, and their biogenesis, cargos, biological functions, and applications. In addition, we discuss the challenges and opportunities in this field.

While nanopore direct RNA sequencing has substantially advanced transcriptomics, its detection of RNA modifications remains primarily focused on abundant biological base modifications. However, therapeutic RNAs employ a diverse catalog of modifications, including base, sugar, and backbone modifications, to enhance stability and pharmacological properties. To address this gap, we systematically evaluated a set of therapeutically relevant modifications [phosphorothioate (PS)], sugar [2′-O-methylation (2′OMe), 2′-Fluoro (2′F), locked nucleic acid (LNA), 2′-O-(2′-methoxyethyl) (2′MOE)], and base [N1-methylpseudouridine (m1Ψ), 5-methylcytidine (m5C), 5-methoxyuridine (5moU), and 5-iodocytidine (5iodoC)] using direct RNA nanopore sequencing. Modifications were systematically analyzed using basecall errors, raw current signals, and modification-aware basecalling models. Ribose modifications, m1Ψ, and 5moU induced significant error rate increases and noticeable current alterations, whereas 2′OMe and 2′MOE affected dwell time adjacent to the pore. In contrast, PS linkages produced only slight current alterations without increasing basecalling errors. We further evaluated modification-aware basecallers for 2′OMe and m5C. While these tools can distinguish modification types, they are limited by poor quantification accuracy and high local error rates, especially for 2′OMe. This study establishes a critical performance baseline, clarifying the current capability and limitations of nanopore technology for the analysis of therapeutically relevant RNA modifications.

Recent advances in in silico protein design and bioinformatics have enabled the rapid generation of candidate sequences for functional proteins. However, experimental validation remains a bottleneck, largely due to time-consuming DNA assembly and cell-based cloning processes. Technologies that reduce the time required to convert synthetic oligonucleotides (oligos) into expressed proteins are therefore of considerable interest. Here, we demonstrate that phase-separated droplets formed by the intrinsically disordered protein (Ddx4N1) concentrate both oligos and ligation enzymes, enabling efficient oligo assembly at nanomolar to sub-nanomolar concentrations that are typically inaccessible to conventional ligation-based methods. The assembled products can be directly introduced into femtoliter-scale microreactors for digital cell-free gene expression, allowing protein expression from single assembled DNA molecules without polymerase chain reaction amplification or cellular cloning. Simultaneous expression of two distinct proteins from separately assembled DNA templates in a one-pot reaction was also demonstrated. The complete workflow—from oligo assembly to detectable protein expression—can be performed within half a day. While further development will be required to enhance reaction parallelization and enable systematic retrieval of sequence information from expressed products, this amplification-free, low-input system establishes a technical foundation for integrating oligo-pool-based gene assembly with digital protein prototyping platforms.

While the impact of non-antibiotic drugs on gut bacteria is well-known, their mechanisms of action remain poorly characterized, and effective mitigation strategies for drug-induced dysbiosis are still limited. Here, we screened bacteria-derived drug-target protein homologs (BDTPHs) mapped to 63 target proteins and 107 associated drugs to quantify “drug–BDTPH–bacterium” interactions. These interactions were validated by co-culture experiments using 10 drugs and 25 strains, enzyme assays, and genetic perturbations in Escherichia coli. Ex vivo and in vivo testing with six drugs showed that over 50% of affected genera exhibited high affinity, indicating microbiota alterations through the “drug–BDTPH–bacterium” axis. Leveraging this quantitative interaction framework, we identified a strain of Bifidobacterium animalis that can competitively bind methotrexate through high-affinity BDTPH, thereby effectively alleviating gut microbiota dysbiosis in vivo. Our findings elucidate a mechanism by which non-antibiotic drug effects on bacterial growth, and suggest a universal homology-based competition strategy to restore drug-disrupted microbiota. Here, the authors uncover a mechanism by which drug-target homologs mediate non-antibiotic drug effects on bacterial growth, and suggest a universal homology-based competition strategy to reverse MTX-induced microbiota dysbiosis.