CRISPR-Cas are widespread adaptive immune systems that protect bacteria and archaea from mobile genetic elements such as bacteriophages. Metagenomic sequencing identified CRISPR-Cas systems in phage genomes; however, their functions remain largely unknown. Here, we present Cas12r-CRISPR, a novel type V CRISPR-Cas system encoded by Lalka phages infecting thermophilic Thermus bacteria. We determined Cas12r-CRISPR PAM consensus sequence and crRNA structure and showed, that when provided with appropriate spacers and expressed in Thermus thermophilus, Cas12r-CRISPR efficiently interferes with plasmid transformation as well as infection by diverse Thermus phages. In the course of Lalka phage infection, the Cas12r-CRISPR locus is expressed with middle phage genes and its transcripts are among the most abundant phage RNAs. Notably, most Cas12r-CRISPR spacers target integrative mobile elements widespread in Thermus genomes. Both Lalka phages and targeted integrative mobile elements use host tRNA genes as attachment sites. We therefore propose that Cas12r-CRISPR participates in an inter-MGEs conflict.

Arsenic (As) contamination in agroecosystems poses significant risks to food security and human health. The mechanisms of As speciation change in soil within wetland rhizospheres are understood, but their location-precise importance within heterogeneous root-associated microbiomes is uncertain. While microbial processes are often considered dominant drivers of As redox transformations, the role of abiotic factors such as reactive oxygen species (ROS) remains underexplored due to limited in situ evidence. Here, we combined multiple high-resolution in situ techniques to map microscale distributions of As(III)/As(V) and key environmental parameters in rice rhizospheres across three paddy soils. A novel ratiometric fluorescent approach was developed for in situ visualization of ROS. Strong spatial correspondence was observed between ROS hotspots and decreased As(III) (R2 = 0.797), exceeding that for O2 (R2 = 0.348). Integration of imaging with functional gene analysis (aioA, arsC), sterilization and ROS-quenching experiments, and structural equation modeling indicates that ROS-driven processes play a crucial role under the studied conditions. However, as gene abundance reflects microbial potential rather than activity, microbial contributions cannot be excluded. These findings highlight ROS as a key regulator of As speciation and provide new insights into coupled abiotic–biotic processes in rhizosphere environments.

Oxford Nanopore Technologies (ONT) rapid library preparation kits use transposase-mediated tagmentation to attach click chemistry functionalized oligonucleotide duplexes to fragmented DNA, followed by click chemistry to conjugate Rapid Adapter (RA) sequencing adapters. A similar protocol is used in 16S rRNA gene amplicon and PCR-amplified rapid whole-genome sequencing workflows. Here, we describe custom oligonucleotides with dibenzocyclooctyne (DBCO) added onto PCR primer 5′ termini. After standard PCR amplification, DBCO-modified amplicons react spontaneously with RA sequencing adapters, producing sequencing-ready libraries in minutes without enzymatic processing. All configurations employ an asymmetric design in which the DBCO modification is restricted to a single primer, leaving the opposite primer available for barcoding at low cost. We validate three primer architectures: (i) direct attachment of DBCO to a target-specific primer, (ii) a universal DBCO-modified oligonucleotide used in a two-step PCR workflow, and (iii) a three-primer single-pot reaction combining the universal DBCO oligonucleotide with unmodified target-specific primers. These configurations are validated using full-length 16S rRNA gene amplicons sequenced on a PromethION flow cell. DBCO-modified primers are synthesized either commercially or in-house via DBCO-TFP ester conjugation to 5′-amino oligonucleotides and remain fully active through standard PCR thermocycling. The best-performing configuration used a two-step PCR with a universal oligonucleotide and achieved higher pore occupation and reads than comparable commercial solutions. This approach reduces library preparation reagent costs compared to available kits, as the initial synthesis cost is lower than existing amplicon sequencing kits, while providing enough material for hundreds or thousands of PCR reactions. This is further applicable to an unlimited number of gene targets beyond 16S sequencing.

Microbial diversity is often assumed to be limited by the number of available resources, yet many communities persist well beyond that expectation. Understanding the mechanisms that enable such coexistence remains a central question in microbial ecology. Here, using a four-species bacterial consortium, we asked whether coexistence can emerge from interactions between species rather than from the external environment alone. Across 31 simple nutrient conditions, including 16 single-resource environments, all four species persisted and repeatedly reached stable coexistence. We then chose 27 additional conditions to further probe the boundaries of coexistence by varying resource concentrations, temporal dynamics, nutrient complexity and relief of auxotrophy-associated dependencies, and only observed the extinction of one species in one of these conditions. Although the community composition in each environment was largely shaped by species' fitness on the supplied resources, experimental assays and consumer-resource modeling showed that the coexistence was not explained by resource supply, but rather by cross-feeding and niche partitioning of metabolic byproducts. These metabolic interactions were strong enough to sustain coexistence even for species unable to use the supplied resources directly. Furthermore, robust coexistence across environments appears to be an emergent property of microbial communities, ingrained in members' metabolic byproduct profiles and niche differences. Our findings demonstrate how microbes can increase the chemical complexity of their environment sufficiently to maintain coexistence well beyond what is expected from external resource supply.

Horizontal gene transfer drives microbial evolution and offers a powerful strategy for precision microbiome engineering. To track gene transfer within complex communities, we previously developed RNA-Addressable Modification (RAM), an RNA-barcoding technology where a mobile catalytic RNA barcodes host 16S ribosomal RNA (rRNA) upon gene transfer. However, the first-generation RAM suffered from low barcoding efficiency and lacked modularity, limiting its sensitivity and versatility. Here, we present RAM v2, a re-engineered system with significantly enhanced performance and modularity. By incorporating natural ribozyme structural motifs and improved barcode stability, we achieved a ~200-fold increase in barcoded rRNA signal. To enhance modularity, we integrated CRISPRi-based repression and ribozyme insulators, facilitating easy promoter swapping. We validated RAM v2 on a mobilisable plasmid delivered to a complex wastewater microbial community, demonstrating a substantial increase in signal over the original system while barcoding similar taxa. These improvements enable higher-resolution, more sensitive monitoring of horizontal gene transfer, providing a robust toolkit for accelerating the study of gene transfer in microbial communities and advancing targeted microbiome engineering.

Modification at specific sites has long been a central goal in peptide and protein chemistry. Introducing functional groups at specific sites is an ideal state for protein function research and the modification of protein properties, as it can eliminate the interference caused by heterogeneous modifications. Recent downstream applications have placed even greater demands on site selectivity. This review examines the latest advances in the site-specific modification of peptides and proteins, organizing the reactions into four categories: exploiting disparities in chemical environments, tag-and-modify approaches, proximity-driven chemical modification, and enzymatic strategies. For each reaction, we detail the underlying design rationale and highlight downstream chemical and biological applications. Collectively, these methods have achieved remarkable gains in terms of specificity, efficiency, and scope, furnishing a versatile chemical toolbox for the site-selective functionalization of peptides and proteins. We conclude by summarizing the current state of the field and outlining prospective directions for future development.

The molecular mechanisms that constrain recombinant protein production and the importance of synonymous codon usage have not been fully elucidated. Codon frequency varies between different organisms, and rare codons are often avoided in “codon-optimized” synthetic gene constructs. Overexpression of tRNA genes is an alternative strategy to compensate for suboptimal codon usage, but the consequences and limitations of this approach have not been widely studied. Here, we develop and characterize a versatile collection of pCODE plasmids for tRNA overexpression in E. coli and show that they complement rare codons with very modest limitations or negative effects, regardless of the position, type, number, or distribution of rare codons in the expressed gene. One pCODE plasmid encodes an inducible tRNA expression cassette and shows potential as a new type of genetic regulation based on inducible rare codon complementation. The pCODE tRNA expression plasmids provide mechanistic insights into synonymous codon usage and add to the molecular biology toolbox that enables recombinant protein production.

Plant-associated microbiomes are central to crop productivity, nutrient efficiency, and stress resilience, yet conventional microbiome manipulation strategies, largely based on microbial inoculation and agronomic management, often suffer from inconsistent field performance and limited persistence. Although several recent reviews have discussed CRISPR-mediated plant–microbe engineering and synthetic microbial community (SynCom) design separately, few reviews integrate genome editing, ecological stability of microbiomes, and climate-resilient agricultural applications within a unified conceptual framework. Recent advances in molecular biotechnology are transforming this landscape by enabling precision engineering of plant-microbe interactions at genetic, metabolic, and community levels. In particular, synthetic biology tools including CRISPR/Cas genome editing, RNA interference, and syncoms, now allow targeted modification of plant traits governing microbial recruitment, microbial pathways underpinning nutrient cycling and stress tolerance, and community-level functional complementarity. This review integrates molecular genetics, microbial ecology, and systems-level microbiome design to frame the plant and its microbiome as an engineerable holobiont. We integrate insights from genome editing in plants and microbes, omics-guided SynCom design, climate-resilience mechanisms, and emerging AI-assisted decision frameworks, including machine learning and ecological modeling approaches used to analyze multi-omics datasets, and predict plant–microbiome interactions across experimental and field-based studies. Importantly, we critically assess limitations related to ecological stability, trait trade-offs, biosafety, and regulatory challenges that constrain large-scale deployment. By bridging genome-enabled microbiome manipulation with ecological design principles, this review proposes an integrative framework for climate-smart microbiome engineering and identifies key research priorities required to transition from empirical inoculation toward predictive, sustainable, and socially responsible agricultural biotechnology.

For much of the past century, carbon dioxide (CO2) has received little attention scientifically outside of its role as a byproduct in the industrialization of the global economy. This trend has recently been upended where, due to mounting environmental concerns, CO2 has been brought squarely into the public consciousness. This surge in activity has contributed to a once unimaginable idea now pervading the scientific community: could CO2, a highly stable byproduct of hydrocarbon combustion, be recycled and converted back into useful chemicals and fuels? Owing to its ubiquitous nature and availability at truly massive quantities, it is thought that CO2-based products could offer a meaningful pathway toward lowering the environmental impact of many of the top industrial products while also enhancing supply chain diversification and resilience. In this manuscript we provide a holistic review of the pathways for CO2 conversion, the underlying chemistry and challenges involved in the transformation to products, and considerations for commercialization.