RMH

Class 1 CRISPR–Cas systems utilize multi-subunit effector ribonucleoprotein complexes to identify and target DNA. Upon recognition, type I systems recruit the helicase/nuclease Cas3 for DNA degradation, while type IV-A systems use the helicase CasDinG for transcriptional repression. Here, we developed two recombinant class 1 CRISPR–Cas genome editing tools for inducing large genomic deletions: the compact type I-Fv (also termed I-F2) system from Shewanella putrefaciens and the type IV-A1 system from Pseudomonas oleovorans. In the latter, CasDinG was engineered to include a C-terminal HNH nuclease domain, conferring DNA cleavage activity and enabling analysis of CasDinG processivity. Whole-genome sequencing of Escherichia coli BL21-AI was used to monitor genome reduction and DNA repair mechanisms in response to CRISPR–Cas-induced damage. Small deletions were flanked by microhomologies, consistent with repair via alternative end joining, whereas deletions larger than 10 kb consistently terminated at nearby IS1 elements, implicating these sequences in the repair process. This study introduces compact type I and engineered type IV-A genome editing tools with distinct protospacer-adjacent motif requirements and provides new insights into CasDinG evolution and the DNA repair pathways engaged during CRISPR–Cas-mediated genome editing.

The Gene Ontology (GO) knowledgebase https://geneontology.org is a comprehensive resource describing the functions of genes. The GO knowledgebase is regularly updated and improved. We describe here the major updates that have been made in the past 3 years. The ontology and annotations have been expanded and revised, particularly in several areas of biology: cellular metabolism, multi-organism interactions (e.g. host-pathogen), extracellular matrix proteins, chromatin remodeling (e.g. the “histone code”), and noncoding RNA functions. We have released version 2 of a comprehensive set of integrated, reviewed annotations for human genes, which we call the “functionome.” We have also dramatically increased the number of GO-CAM models, with over 1500 models of metabolic and signaling pathways, primarily in human, mouse, budding and fission yeast, and fruit fly. Finally, we discuss our current recommendations and future prospects of AI in the use and development of GO.

Colouration of textiles is responsible for 3% of global greenhouse gas emissions and 20% of global wastewater generation. An estimated 20% of the dyes and pigments used in textile colouration is lost to wastewater. Synthetic dyes and pigments, along with many of the chemicals added to their formulations, are

nonbiodegradable, persistent, and bioaccumulative in aquatic

ecosystems, posing signi cant risks throughout the food chain. Regulatory bodies in both Europe and the United States are continuing to ban, restrict, and phase out many synthetic dyes and pigments. The EU recently banned a significant number of common textile dyes known to release carcinogenic compounds,

while in the United States, 8 common synthetic food dyes are

being targeted for immediate ban in the US food supply, with the FDA planning to phase out all remaining synthetic food dyes. While bio-based alternatives are available on the market, they are

currently more expensive, restricting their application to niche product applications that can absorb the price premium. The total market size for dyes and pigments is more than US$ 50 billion and is growing at a rate of 3–10% annually. The ‘cost of being unsustainable’ is increasing rapidly, particularly in the EU, as new regulations make the use of hazardous chemicals more difficult

and expensive. This is driving a shift toward the use of more sustainable chemicals.

Base editors enable precise genome modification but are constrained by bystander edits that limit their applicability. Existing strategies to enhance precision often compromise efficiency and remain highly sequence dependent. Here we present a parallel engineering approach that optimizes both guide RNAs and the deaminase enzyme to minimize bystander editing without sacrificing activity. We designed a library of 3′-extended guide RNAs and identified context-dependent variants that improved specificity. Using a precision-driven phage-assisted evolution system and protein language models, we evolved adenine base editor variants two- to threefold more precise than adenine base editor ABE8e while maintaining high efficiency across a library of thousands of human pathogenic contexts in vitro. Our findings establish a scalable framework for precision engineering of base editors, addressing a major challenge in genome editing. Base editors are made more precise through deaminase and gRNA engineering.

Prime editing (PE) enables the precise installation of intended base substitutions, small deletions or small insertions into the genome of living cells. While the use of Cas9 nickase can avoid DNA double-strand breaks (DSB), undesired insertions and deletions (indels) often accompany the correct edits, particularly when PE activity increased. Here we show that the anti-CRISPR (Acr) protein AcrIIA5 can significantly enhance PE activity by up to 8.2-fold while markedly reducing byproduct indels. Further investigation reveals that AcrIIA5 can promote PE across various approaches (PE2, PE3, PE4, PE5, and PE6), edit types (substitutions, insertions and deletions), and endogenous loci. Mechanistically, AcrIIA5 appears to inhibit the re-nicking activity of PE complex rather than enhancing the core editing machinery itself, suggesting a distinct mode of interaction with Cas9. Overall, we demonstrate that a known “inhibitor” Acr protein can unexpectedly acting as an “enhancer” of CRISPR/Cas-based genome editing, providing an effective strategy to optimize PE specificity and activity. Prime editing enables precise genetic modification but often suffers from unwanted byproducts. Here, authors show that the anti-CRISPR protein AcrIIA5 unexpectedly enhances prime editing efficiency while reducing unintended indels, offering an effective strategy to improve activity and specificity.

Directed evolution facilitates functional adaptations through stepwise changes in sequence that alter protein structure. While most campaigns yield solutions that maintain the framework of a rigid protein architecture, a few have produced enzymes with more notable structural differences. One example is a polymerase that was evolved to synthesize threose nucleic acid (TNA) with near-natural activity. Understanding how this enzyme arose provides a model for studying pathways that guide enzymes toward more productive regions of the fitness landscape. Here, we trace the evolutionary trajectory of an unnatural polymerase by solving crystal structures of key intermediates along the pathway and evaluating their biochemical activity. Contrary to the view that fidelity is a product of increased catalytic efficiency, we find that accuracy and catalysis are decoupled activities guided by separate ground-state and transition-state discrimination events. Together, these results offer a glimpse into the forces responsible for shaping the emergence of new enzyme functions. Engineering polymerases to synthesize alternative genetic polymers remains a challenging problem in synthetic biology. The current study offers insights into the structural and biochemical changes responsible for improving the fidelity and catalytic activity of a laboratory evolved TNA polymerase.

Extracting fungal hyphae with their naturally associated microbiota from soil samples presents a significant challenge due to their small size, typically in the micrometer range, and the formation of dynamic fungal networks. We combined elements of previous protocols and automated the wet-sieving steps of the methodology to efficiently extract fungal hyphae from various soil types, including natural loamy soils. This approach reduces manual handling, minimizes operator-dependent variability, and shortens processing time by up to 2.5-fold. Unlike earlier methods that require sand or glass bead supplementation, which can introduce artificial conditions and limit large-scale field applications, our Sieving and Sucrose Centrifugation (SSC) method avoids these drawbacks. The SSC technique enables both quantification of hyphal length density (HLD) and, importantly, preserves surface-associated microbes for downstream analyses. Among the tested methods, SSC yielded the highest hyphal length density. Using a combination of microscopy, molecular techniques, and next-generation sequencing (NGS), we demonstrate that this method allows targeted study of bacteria tightly attached to fungal hyphae. Furthermore, the SSC approach effectively enriched fungal hyphae from a highly diverse soil community, establishing a dependable tool for advancing research on fungal hyphae as microbial hotspots in soil ecosystems.