Nanopores have become transformative tools in single-molecule chemical analysis, enabling detailed interrogation of molecular interactions and reaction dynamics. These advancements have revolutionized the characterization of chemical kinetics and stereospecificity, broadening nanopore applications. This review evaluates the principles of nanopore single-molecule chemistry, highlighting breakthroughs in chemically reactive nanopore construction via site-specific mutagenesis, semisynthetic engineering, and orthogonal modifications. Notably, we highlight the innovative strategies enabling precise subunit stoichiometry control to ensure single-molecule reactions, and the integration of machine learning for high-fidelity ionic current analysis. These developments position nanopores as versatile tools for intricate molecular detection in fundamental and applied research. Looking forward, nanopore single-molecule chemistry promises an impact on diagnostics, environmental monitoring, and precision medicine. Integration of molecular dynamics simulations, artificial intelligence–driven protein design frameworks, and microsystems technology may expand detectable species, enhancing robustness and lowering detection limits. Such advancements will deepen our understanding of chemical transformations and support meaningful real-world applications of nanopore technologies.

Pseudomonadota (formerly Proteobacteria) are prevalent in the commensal human gut microbiota, but also include many pathogens that rely on secretion systems to support pathogenicity by injecting proteins into host cells. Here we show that 80% of Pseudomonadota from healthy gut microbiomes also have intact type III secretion systems (T3SS). Candidate effectors predicted by machine learning display sequence and structural features that are distinct from those of pathogen effectors. Towards a systems-level functional understanding, we experimentally constructed a protein–protein meta-interactome map between human proteins and commensal effectors. Network analyses uncovered that effector-targeted neighborhoods are enriched for genetic variation linked to microbiome-associated conditions, including autoimmune and metabolic diseases. Metagenomic analysis revealed effector enrichment in Crohn’s disease but depletion in ulcerative colitis. Functionally, commensal effectors can translocate into human cells and modulate NF-κB signalling and cytokine secretion in vitro. Our findings indicate that T3SS contribute to microorganism–host cohabitation and that effector–host protein interactions may represent an underappreciated route by which commensal gut microbiota influences health. Large-scale computational and in vitro analyses identify commensal type III secretion systems and substrates in the human gut microbiome that can interact with human proteins to modulate immune pathways.

Single-chain variable fragments (scFvs) antibodies are used in diagnostic and therapeutic biopharmaceuticals. However, its production is limited due to insoluble expression and lack of a universal purification ligand. Here, we report a novel two-in-one protein tag, termed CSQ-tag, utilizing a calsequestrin (CSQ) protein to enable the soluble expression and calcium-dependent purification for scFvs. The CSQ-tag enhanced the solubility of four different therapeutic scFvs with an average solubility of 83.8 ± 4.6% in the BL21(DE3) strain, despite its reducing cytoplasmic environment. Compared to other tags, CSQ-tag showed superior solubility, with 1.8-fold higher solubility than MBP-tag across four scFvs. The solubility enhancement of the CSQ-tag is attributed to two main characteristics: highly acidic sequences with a low isoelectric point of 3.95, and intrinsically disordered protein properties. Furthermore, CSQ-tag can cost-effectively purify therapeutic scFvs with high purity over 95% using the calcium-dependent phase transition properties. Importantly, the anti-VEGF-scFv produced with the CSQ-tag showed a binding affinity of 25.8 pM, similar to that of the existing therapeutic brolucizumab. Therefore, CSQ-tag can significantly improve productivity and simultaneously maintain scFvs’ functional integrity, thereby serving as a promising tool for universal expression and purification of therapeutic scFvs. A calsequestrin-based protein tag enhances the soluble expression of single-chain antibody fragments and enables their calcium-dependent purification, offering a cost-effective platform for therapeutic antibody production.

CRISPR–Cas systems are transformative tools for gene editing that can be tuned or controlled by anti-CRISPRs (Acrs)—phage-derived inhibitors that regulate CRISPR–Cas activity. However, Acrs that can inhibit biotechnologically relevant CRISPR systems are relatively rare and challenging to discover. To overcome this limitation, we describe a highly successful and rapid approach that leverages de novo protein design to develop new-to-nature proteins for controlling CRISPR–Cas activity. Here, using Leptotrichia buccalis CRISPR–Cas13a as a representative example, we demonstrate that Acrs designed using artificial intelligence (AIcrs) are capable of highly potent and specific inhibition of CRISPR–Cas13a nuclease activity. We present a comprehensive workflow for design validation and demonstrate AIcr functionality in controlling CRISPR–Cas13 activity in bacterial and human cells. The ability to design bespoke inhibitors of Cas effectors will contribute to the ongoing development of CRISPR–Cas tools in diverse applications across research, medicine, agriculture and microbiology. Taveneau et al. leverage artificial-intelligence-driven protein design to create inhibitors that control RNA-targeting enzymes in cells, revealing a strategy to rapidly design off-switches for RNA-editing systems.

Guide RNA (gRNA) arrays can enable targeting multiple genomic loci simultaneously using CRISPR-Cas9. In this study, we present a streamlined and efficient method to rapidly construct gRNA arrays with up to 10 gRNA units in a single day. We demonstrate that gRNA arrays maintain robust functional activity across all positions, and can incorporate libraries of gRNAs, combining scalability and multiplexing. Our approach will streamline combinatorial perturbation research by enabling the economical and rapid construction, testing, and iteration of gRNA arrays. To facilitate the adoption of this approach, we have made a web tool to design oligo sequences necessary to assemble gRNA arrays.

Gene expression is dynamically regulated by gene regulatory networks comprising multiple regulatory components to mediate cellular functions. An ideal tool for analyzing these processes would track multiple-component dynamics with both spatiotemporal resolution and scalability within the same cells, a capability not yet achieved. Here, we present CytoTape, a genetically encoded, modular protein tape recorder for multiplexed and spatiotemporally scalable recording of gene regulation dynamics continuously for up to three weeks, physiologically compatible, with single-cell, minutes-scale resolution. CytoTape employs a flexible, thread-like, elongating intracellular protein self-assembly engineered via computationally assisted rational design, built on earlier XRI technology. We demonstrated its utility across multiple mammalian cell types, achieving simultaneous recording of five transcription factor activities and gene transcriptional activities. CytoTape reveals that divergent transcriptional trajectories correlate with transcriptional history and signal integration, and that distinct immediate early genes (IEGs) exhibit complex temporal correlations within single cells. We further extended CytoTape into CytoTape-vivo for scalable, spatiotemporally resolved single-cell recording in the living brain, enabling simultaneous weeks-long recording of doxycycline- and IEG promoter-dependent gene expression histories across up to 14,123 neurons spanning multiple brain regions per mouse. Together, the CytoTape toolkit establishes a versatile platform for scalable and multiplexed analysis of cell physiological processes in vitro and in vivo.

Bacterial antiphage defense systems play essential roles in microbial ecology, yet their dynamics within urban wastewater systems (UWS) remain poorly characterized. Here, we performed comprehensive metagenomic and plasmidome analyses on 78 wastewater samples collected during two seasons and four sampling points across UWS from three European countries. We observed a significant reduction in the abundance, diversity, and mobility potential of defense systems during biological treatment. However, these reductions were not directly correlated with changes in microbial abundance. Defense systems were significantly enriched on plasmids, particularly conjugative plasmids, where their gene density was approximately twice as high as on chromosomes and remained relatively stable across compartments. In contrast to chromosomal defense systems, plasmid-borne systems exhibited more frequent co-localization with a wide range of mobile genetic elements (MGEs)-associated genes, thereby facilitating multilayered dissemination networks. Furthermore, we detected a strong correlation between phage abundance and host defense system profiles, indicating ongoing phage-host co-evolutionary dynamics in these environments. In sum, our results demonstrate that UWS reduce the abundance and diversity of bacterial defense system genes. However, plasmid-associated defense systems can persist through shared mobile genetic reservoirs. These findings underscore the critical role of plasmids in bacterial immunity and provide new insights into defense system dynamics within urban wastewater environments.

Assessing the relative roles of chance and necessity in evolution is of wide interest, but it requires evolving the same population under the same environment multiple times—a virtually impossible task in nature that has been repeatedly accomplished in the laboratory. Capitalizing on the transcriptome data collected in 10 laboratory evolution studies conducted in 22 distinct environments, we investigate the evolutionary repeatability of a total of 182,103 gene expression traits in a prokaryotic and five eukaryotic species. The number of gene expression traits exhibiting concordant changes in direction and magnitude between two replicate populations typically exceeds the chance expectation by 10 to 100 standard deviations. Contrasting replicate evolution in the same environment with that in different environments suggests that the concordance in expression evolution is largely attributable to environment-specific selections, a finding that is further supported by comparing with the outcome of mutation accumulation experiments where the efficacy of selection is minimized. Additionally, genes controlled by more transcription factors tend to show more repeatable expression evolution, likely due to a higher certainty in the occurrence of mutations altering their expressions. In conclusion, contrary to almost unrepeatable genotypic evolution, phenotypic evolution during environmental adaptation is quite repeatable and deterministic, at least for gene expressions. Experimental evolution enables evaluation of the relative roles of chance and necessity in evolution. This study compiles transcriptomic data from experimental evolution of a prokaryotic and five eukaryotic species in 22 environments to reveal that gene expression evolution is often repeatable and deterministic.

The leather industry is at a pivotal moment on its path towards sustainability. Although many bio-based leather alternatives can reduce environmental impact, we need clear definitions of such alternatives to guide materials innovations that truly bring them into the mainstream, sustainable market, as highlighted in a recent court ruling.