CRISPR-Cas systems often rely on collateral cleavage of nucleic-acid substrates to combat recognized mobile genetic elements. Of the CRISPR-associated (Cas) RNA-guided effector nucleases, Cas12a2 stands out as the only known example exhibiting rapid collateral cleavage of three distinct substrates: single-stranded (ss)RNA, ssDNA, and double-stranded (ds)DNA, after activating upon binding cognate RNA. However, little is known about the underlying mechanisms of collateral cleavage. Here, we show, using enzyme kinetics and inhibition assays, that Cas12a2 preferentially cleaves collateral DNA over RNA substrates, even when RNA substrates are more abundant. Additionally, using enzyme mutants, enzyme kinetics, and plasmid cleavage assays, we determine that the dsDNA cleavage mechanism relies on the ‘aromatic clamp’ residues that stabilize unwound and distorted dsDNA in the RuvC nuclease active site. Leveraging the cleavage preference for collateral DNA, we demonstrate that RNA-activated Cas12a2 can readily cleave a ssDNA probe in the presence of high concentrations of non-target RNA, while an RNA-targeting Cas13a cannot. This work provides foundational kinetic and biochemical insights into the collateral cleavage mechanism and substrate preferences of Cas12a2, with immediate implications for understanding Cas12a2-based immunity and developing Cas12a2-based technologies.

Nanopores have been explored as a potential platform for protein analysis following the success of nanopore nucleic acid sequencing. However, protein sequencing remains technically challenging and has not yet been established for proteomics use. Here a nickel-immobilized Mycobacterium smegmatis porin A (MspA-NTA-Ni) nanopore is shown to enable the identification of a range of proteomic analytes, including amino acids and peptides up to 39 amino acids in length. Under identical conditions, signals corresponding to 20 proteinogenic amino acids, 4 post-translationally modified amino acids, 32 peptides, 6 modified peptides, 11 bioactive peptides and 2 neoantigen peptides were recorded. Machine-learning-based analysis enabled classification of these analytes with a validation accuracy of up to 97.4% within the studied dataset. The MspA-NTA-Ni nanopore supports both direct peptide identification and peptide profiling following enzymatic hydrolysis. As a proof of concept, a reference peptide was digested using exo- and endopeptidases to generate overlapping peptide fragments. Nanopore measurements combined with machine learning predictions enabled the identification of fragment compositions and partial sequences, allowing reconstruction of the original peptide sequence. This hydrolysis-based approach shows sensitivity to sequence alterations, including mutations, deletions and post-translational modifications, indicating potential utility for targeted peptide characterization. A nickel-modified nanopore enables simultaneous, unambiguous identification of amino acids and peptides. Direct analysis or hydrolysis-based sequencing can be used for profiling and reconstruction, and detect single-residue mutations and post-translational modifications.

Muconic acid (MA) is characterized by two reactive carboxylic acid groups and two conjugated double bonds, making it a highly valuable industrial platform chemical with significant market potential. It serves as a key intermediate in the manufacturing of important commercial chemical products such as adipic acid and terephthalic acid. The finite fossil-based resources and climate issues due to CO2 emission have necessitated the microbial routes for the production of MA, a potential alternative to fossil-based synthesis. This review provides a comprehensive overview of recent progress in the biological production of MA. The article begins with an outline of the present catalytic routes and known biochemical pathways for MA biosynthesis. The review then focuses on metabolic engineering strategies employed in various microbial hosts including E. coli, Corynebacterium glutamicum, and Pseudomonas putida to enhance MA production from diverse feedstocks such as sugars, aromatic compounds, and lignin-derived substrates. Special attention is given to pathway optimization, host tolerance, and strategies enabling efficient conversion of lignin-derived intermediates within integrated biorefinery frameworks. Key challenges associated with scaling up bio-based MA production to industrial levels are discussed, along with potential strategies for developing robust and efficient microbial cell factories. The review concludes with future perspectives and recommendations to accelerate research progress and development in this field.

Water scarcity and heat stress are major challenges to sustainable agriculture and global food security. Here we present a hierarchically structured living material composed of directionally guided mycelium fibres, designed to regulate soil moisture and thermal load by harvesting atmospheric water and dissipating heat. The material is fabricated by cultivating Pleurotus ostreatus on a cellulose scaffold under asymmetric gas exposure, which directs hyphal growth into a vertically organized architecture with a fused cellulose–mycelium composite below and a porous aerial mycelium network above. This configuration creates a built-in wettability gradient. Aerial mycelium fibres become hydrophobic through the self-assembly of hydrophobins during upward growth into the air, whereas cellulose fibres and submerged mycelium remain hydrophilic. This gradient enables directional water transport, driving condensed atmospheric moisture from the aerial surface into the soil. Simultaneously, the porous aerial network back-scatters solar radiation and emits thermal radiation, lowering the surface temperature and promoting water vapour condensation. Field trials show that this biologically grown soil envelope increases tomato yield by approximately 28% (wet weight) compared to bare soil. Overall, this work demonstrates how the guided biomanufacturing of living materials can be harnessed to create functional, scalable and sustainable solutions for climate-resilient agriculture. This study introduces a scalable, hierarchically structured living mycelium–cellulose material that passively harvests atmospheric water and dissipates heat to regulate soil moisture and temperature, as demonstrated by increased crop yields in field trials.

Root exudates comprise a diverse mixture of non-polar and amphiphilic compounds that are only partially recovered by aqueous extraction methods, yet can rival polar metabolites as carbon sources for microorganisms. Because many quorum-sensing (QS) signals are fatty-acyl derivatives, lipid-rich microhabitats at the root–soil interface are likely to influence signal partitioning, persistence, and local QS thresholds. We propose a lipid-mediated framework in which plant-derived lipids modulate QS through four nodes: receptor mimicry/antagonism, quorum quenching, membrane/microenvironment regulation, and lipid-dependent resource gating. These mechanisms operate across two lipid-rich interfaces - the rhizosphere and the arbuscular mycorrhizal fungi (AM fungi) hyphosphere - where host lipid fluxes may restructure microbial community composition. Combined with QS-sensitive changes in root exudation, this spatially structured lipid circuit could generate feedbacks influencing microbiome composition and function, with potential implications for microbiome engineering.

Prime editing is a versatile clinical genome editing method that enables precise substitutions, small insertions and deletions at specified locations in the genomes of living systems including human cells. Although non-viral lipid nanoparticle (LNP) delivery of RNA in vivo has become a preferred method for gene editing in animals and patients, its application to complex, three-component prime editing systems has yielded low editing efficiencies. Here we developed a systematic prime editing LNP (PE-LNP) optimization platform that addresses key bottlenecks in cargo design that limit editing efficiency. This generalizable workflow yielded PE-LNPs that can achieve 49% average in vivo prime editing in the bulk mouse liver with a single dose of 2 mg kg−1. We applied our workflow to the correction of PAH R408W, a cause of phenylketonuria, in a mouse model and achieved prime editing efficiencies and serum phenylalanine levels anticipated to be curative. We also show that PE-LNPs minimize off-target editing compared with DNA delivery methods, induce only transient elevation of liver enzymes and can be dosed repeatedly to improve editing efficiencies. These PE-LNP systems provide an attractive alternative to viral delivery by offering transient expression that minimizes off-target editing, no observed long-term toxicity and high levels of non-viral in vivo liver prime editing. A systematic workflow is used to optimize lipid nanoparticle-based prime editing systems that enable efficient editing and disease correction in human cells and mouse tissue, and overcome key limitations of transient delivery.