Nucleotide sequences in the FASTQ or BAM format are widely shared, yet derived from platform-specific raw data outputs that differ across sequencing platforms. In Oxford Nanopore Technologies (ONT) sequencing, raw signal data contain valuable biological information and enable basecaller optimization and modification detection. These raw signals also underpin algorithms that could improve ONT device portability and enhance target enrichment efficiency through adaptive sampling. Nevertheless, the storage and sharing of raw nanopore data remain limited due to technical constraints and the lack of standardized and centralized infrastructure. To address this challenge, we developed SquiDBase (https://squidbase.org), a dedicated repository for raw microbial nanopore sequencing data with linked processed data and metadata. To maximize immediate utility, we built SquiDPipe, a Nextflow pipeline for the automated removal of human reads from raw nanopore data, sequenced 24 clinically relevant viruses and incorporated them into SquiDBase, and added publicly available reference datasets and new community contributions. By offering a centralized, open-access raw data collection platform, SquiDBase facilitates data sharing, enhances reproducibility, and supports the development and benchmarking of computational tools, reinforcing open science in nanopore sequencing.

Cold seeps host diverse microbes and viruses with numerous unexplored defense and anti-defense systems. Analysis of 3813 microbial and 13,336 viral genomes from 191 metagenomes across 17 cold seep sites reveals extensive microbial defense repertoires, with over 60% representing candidate systems. Experimental validation confirms that several candidates protect against viral infection. These defense systems frequently co-occur, suggesting potential synergistic interactions, and are broadly distributed across sediments. In response, viruses have evolved diverse anti-defense genes, and the concurrent presence of multiple viral and microbial systems highlights intricate coevolution. Functionally critical lineages, such as anaerobic methanotrophic archaea, sulfate-reducing bacteria, and diazotrophs, appear to modify their defensive strategies under ecological and environmental pressures; for example, sulfate-reducing bacteria harbor multiple Gabija systems while corresponding viruses carry anti-Gabija genes, illustrating specific coevolutionary adaptations. Overall, these findings underscore the critical role of virus-microbe interactions in shaping microbial metabolic functions and environmental adaptation in deep-sea ecosystems. Deep-sea cold seeps harbor diverse microbial and viral communities engaged in molecular conflicts. This study reveals extensive and coordinated defense and anti-defense systems that coevolve and shape microbial–virus relationships in this extreme environment.

The messenger RNA 3' untranslated region contains important regulatory sequences, including upstream sequence elements (USEs), which regulate gene expression. One well-characterised USE in the 3'UTR of the Drosophila polo gene affects adult fly phenotypes when disrupted. We have now identified a highly conserved sequence within this USE (DplUSE) in the 3'UTR of several vertebrate genes, including in zebrafish, mouse, and human genomes and show that DplUSE enhances gene expression in human cells and zebrafish embryos. We show that, in humans, DplUSE-containing genes are associated with congenital disease processes, and that disruption of DplUSE function impairs zebrafish development. We also found that HuR/ELAVL1, hnRNPC, and PTBP1/hnRNPI bind to DplUSE RNA and are required for its activity in a human cell line, suggesting a highly conserved mechanism across distantly related species. Our results indicate that PTBP1 has a global function in alternative polyadenylation, activating the selection of distal polyA sites and repressing intronic polyadenylation in DplUSE-containing genes while hnRNPC and HuR modulate their expression. Additionally, we found that a colon cancer-associated SNP in the POU2AF2/C11orf53 3'UTR creates an ectopic DplUSE site, increasing gene expression in zebrafish gut cells and in a human cell line. We have therefore identified a short 3'UTR motif present in diverse vertebrate genes that controls their expression through conserved RBPs interactions and is implicated in human disease.

The expansion of the genetic alphabet through the development of unnatural base pairs (UBPs) has the potential to revolutionize synthetic biology and biotechnology. However, the replication of the UBPs by eukaryotic DNA polymerases is largely unexplored and the sequencing of the UBPs remains challenging. Herein, we explored and demonstrated the activity of human DNA polymerase β (Pol β) for the efficient and specific synthesis and extension of a panel of representative UBPs, including dNaM–dTPT3, dCNMO–dTPT3, and their functionalized derivatives. Based on this, we established a method for the sequencing of DNAs containing different unnatural bases, involving stalled primer extension mediated by Pol β, selective conversion of an unnatural nucleotide into two different natural ones in parallel and further primer extension mediated by Taq DNA polymerase, and Sanger or deep sequencing of the produced natural DNAs to locate the unnatural bases. The precision, universality, and potential for high-throughput applications of this method were demonstrated by the successful sequencing of various DNAs containing one or multiple of different unnatural bases. This work suggests the possibility of integrating the UBPs into the eukaryotic DNA replication systems and provides a technical foundation for the robust sequencing of DNAs with an expanded genetic alphabet.

Nitazenes are an emergent class of synthetic opioids that often rival or exceed fentanyl in their potency. These compounds have been detected internationally in illicit drugs and are the cause of increasing numbers of hospitalizations and overdoses. New analogs are consistently released, making detection challenging — new ways of testing a wide range of nitazenes and their metabolic products are urgently needed. Here, we develop a computational protocol to redesign the plant abscisic acid receptor PYR1 to bind diverse nitazenes and maintain its dynamic transduction mechanism. The best design has a low nanomolar limit of detection in vitro against nitazene and menitazene. Deep mutational scanning yielded sensors able to recognize a range of clinically relevant nitazenes and the common metabolic byproduct in a complex biological matrix with limited cross-specificity against unrelated opioids. Application of protein design tools on privileged receptors like PYR1 may yield general sensors for a wide range of applications in vitro and in vivo. Nitazenes are potent synthetic opioids that are difficult to detect. Here, authors computationally redesign a plant receptor to create sensitive sensors capable of detecting diverse nitazenes and their metabolites in biological samples.

Aspergillus niger serves as a cell factory for efficient enzyme production. The lipase derived from Thermomyces lanuginosus (TLL) is known for its remarkable thermal stability and is extensively utilized in various industrial fields. In this study, a heterologous expression strain, ΔAnTll-11, of TLL was successfully constructed in A. niger. Through fermentation optimization, the lipase activity was enhanced 8.7-fold to 4547.95 U/mL, compared with the initial value of 520 U/mL. Enzymatic characterization indicated that the recombinant lipase exhibited optimum activity at pH 9.5 and 45 °C, and its activity was positively influenced by Ca2⁺, Ag⁺, Mg2⁺, and Cu2⁺. The ΔAnTll-11 strain exhibited enhanced tolerance to high osmolarity, oxidative stress, and thermal shock. Furthermore, transcriptomic analysis of the ΔAnTll-11 strain revealed that half of the total annotated genes (7199 genes) were differentially expressed genes (DEGs). According to protein‒protein interaction network and weighted gene coexpression network analyses, genes involved in ribosome function, amino acid metabolism, glycosylation, energy metabolism, and the MAPK signalling pathway may affect the expression of lipase. The transcriptomic findings elucidated the regulatory mechanisms by which A. niger expresses foreign proteins and enzymes, establishing a groundwork for further enhancing A. niger as a cell factory for efficient enzyme and protein production.

Precise modeling of transcriptional regulation is essential for the rational design of genetic circuits in synthetic biology. Current computational approaches for predicting transcriptional activity (ITX) typically lack mechanistic clarity, composability, and scalability, and require extensive training data. Here, we present a modular thermodynamic modeling framework that explicitly parameterizes molecular interactions among promoters, RNA polymerase (RNAP) and transcription factors (TFs). Implemented as the computational platform, T-Pro, this approach provides robust interpretability, scalability, and predictive power. Experimental validation across three distinct bacteria—Escherichia coli, Bacillus subtilis, and Corynebacterium glutamicum—demonstrates substantial improvements (up to 20-fold) in a composite transcriptional performance metric (Fmax*FC), achieved within only three Design–Build–Test–Learn cycles and fewer than five genetic constructs in total. Furthermore, we validate the framework by engineering multispecies bacterial communication circuit, highlighting its broad utility and generalizability. The principles and tools developed here thus enable efficient, rational optimization of transcriptional regulation across diverse prokaryotic hosts.

We present a molecular strategy that enables the programmable activation of the CRISPR–Cas12a system in response to triplex DNA formation triggered by single-stranded DNA (ssDNA) or RNA inputs. Our triplex-controlled Cas12a assay leverages the high specificity of clamp-like triplex structures to control a toehold-based strand displacement reaction within a rationally designed DNA hairpin (PAM-Switch). Upon displacement and protospacer adjacent motif (PAM) complementation, the Cas12a ribonucleoprotein (RNP) is activated, initiating trans-cleavage and producing a concentration-dependent fluorescent signal. By decoupling target recognition (via triplex formation) from direct hybridization with the Cas12a–crRNA complex, the assay eliminates the need for target-specific crRNAs. This design also allows multiple detection of distinct nucleic acid (NA) targets using a single Cas12a reaction mix. Through the use of triplex-based clamps, the proposed platform achieves enhanced specificity for single-nucleotide variants and supports the detection of both ssDNA and RNA targets across a broad range of lengths (10–20 nucleotides), addressing key limitations in current Cas12a-based diagnostics and opening new avenues for NA sensing.