Immune matching and rejection pose major hurdles in tissue transplantation. Here, we profile HLA-A , HLA-B , and HLA-C alleles in 3,496 Lithuanian donors genotyped at three-field resolution. The five most frequent alleles constitute 74.6% of HLA-A , 43.2% of HLA-B , and 59.2% of HLA-C , with HLA-A*02:01:01, HLA-B*07:02:01, and HLA-C*07:02:01 being the most common. Lithuanian allele frequencies closely resemble those of populations with pre-Neolithic hunter-gatherer ancestry, such as European-American and British groups. We identified 153 double homozygotes and 51 triple homozygotes for HLA-A , HLA-B , and HLA-C . Compatibility modeling showed triple homozygous profiles match 60.5% of Lithuanians (33.3% for double homozygotes), 13.4% of British population, and 7.4% of European-Americans. CRISPR-Cas9 guide RNA design yielded 54 candidates predicted to disrupt HLA-A or HLA-B , while preserving HLA-C , producing edited profiles matching over 98.1% of Lithuanians, 95.8% of European-Americans, and 95.6% of British population. Finally, we established 16 fibroblast lines from double and triple homozygotes, offering a resource for immune-compatibility studies and regenerative medicine applications.

RNA splicing is fundamental to cellular function, yet systematic investigation of its complex regulation has been limited by existing methods. Here, we present SPLiCR-seq ( SPL icing regulator identification through CR ISPR screening), a high-throughput CRISPR screening platform that enables direct measurement of RNA splicing outcomes for pooled genetic perturbations, overcoming limitations of traditional fluorescence-based approaches. Applying SPLiCR-seq to investigate XBP1 splicing during the unfolded protein response (UPR), we conducted targeted and genome-wide screens across diverse cellular contexts, revealing both common and cell-type specific regulators. Notably, we identified GADD34 ( PPP1R15A ) as a novel modulator of IRE1-XBP1 signaling, demonstrating that it directly interacts with IRE1 and functions independently of its canonical role in eIF2α dephosphorylation. Pharmacological inhibition of GADD34 using Sephin1 effectively suppressed XBP1 splicing and alleviated CAR-T cell exhaustion in an ex vivo model, leading to enhanced tumor-killing capacity across multiple cancer models. This work not only establishes a powerful new tool for systematically studying RNA splicing regulation but also uncovers a promising therapeutic strategy for improving CAR-T cell immunotherapy through modulation of the IRE1-XBP1 pathway.

Objective Genome-wide association studies (GWAS) have identified numerous single nucleotide polymorphisms (SNPs) associated with juvenile idiopathic arthritis (JIA), the majority of which are located in non-coding regions such as enhancers. This presents a challenge for pinpointing causal variants and their target genes. Interpreting these loci requires functional genomics data from disease-relevant tissues, which has been lacking for JIA. This study seeks to fill that gap and elucidate the biological mechanisms underlying JIA susceptibility. Methods We performed low-input whole genome promoter Capture Hi-C (PCHi-C) and ATAC-seq on CD4+ T cells from three JIA oligoarthritis patients. To link JIA-associated SNPs to potential causal genes, we integrated PCHi-C data with JIA GWAS summary statistics using our Bayesian prioritisation algorithm, Capture Hi-C Omnibus Gene Score (COGS). ATAC-seq was used to further annotate JIA GWAS loci in CD4+ T cells. We then employed CRISPR activation and interference (CRISPRa/i) in Jurkat cells to validate the prioritised SNPs and their corresponding genes. Results Chromatin interactions between JIA-associated SNPs and gene promoters were identified in 19 of 44 non-MHC JIA loci, linking 61 known and novel target genes to the disease. Through COGS, we prioritised seven putative causal genes for JIA: RGS14, ERAP2, HIPK1, CCR4, CCRL2, CCR2 , and CCR3 . SNPs within promoter-interacting regions (PIRs) of these genes were further validated using CRISPRa/i to confirm their roles in regulating gene expression. Conclusions This study provides insights into the genetic architecture of JIA by integrating genomic and epigenomic data, identifying disease-related genes, functionally validating risk SNPs, and highlighting candidate drugs for repurposing. Key messages What is already known on this topic Recent genome-wide association studies in JIA have identified genetic loci associated with disease risk. However, the precise mechanisms by which these variants contribute to disease pathology remain unclear, as most do not directly alter protein-coding genes. It has been proposed that non-coding SNPs can affect genes that are important in disease through disruption of enhancer-mediated regulatory mechanisms that control their expression, with enhancers exerting their effects through chromatin interactions. Functional characterisation of risk loci is essential to delineate causal SNPs and target genes in JIA. What this study adds This study is the first to utilise low-input Promoter Capture Hi-C to map long-range chromatin interactions in CD4+ T cells from JIA patients, alongside ATAC-seq to assess chromatin accessibility within the same samples. It identifies 61 potential target genes at JIA-associated loci and validates the regulatory roles of some of these through CRISPR activation and interference. This work enhances our understanding of how genetic variants modulate gene expression in immune cells, shedding light on key pathways involved in JIA pathogenesis. How this study might affect research, practice or policy Highlights new potential causal genes in JIA which can help understand the pathological mechanisms in JIA, and suggests the potential to repurpose CCR2/CCR5 inhibitors in JIA.

Metastasis remains the leading cause of cancer-related mortality, yet predicting future metastasis is a major clinical challenge due to the lack of validated biomarkers and effective assessment methods. Here, we present EmitGCL, a deep-learning framework that accurately predicts future metastasis and its corresponding biomarkers. Based on a comprehensive benchmarking comparison, EmitGCL outperformed other computational tools across six cancer types from seven cohorts of patients with superior sensitivity and specificity. It captured occult metastatic cells in a patient with a lymph node-negative breast cancer, who was declared to have no evidence of disease by conventional imaging methods but was later confirmed to have a metastatic disease. Notably, EmitGCL identified HSP90AA1 and HSP90AB1 as predictable biomarkers for future breast cancer metastasis, which was validated across five independent cohorts of patients (n=420). Furthermore, we demonstrated YY1 transcription factor as a key driver of breast cancer metastasis which was validated through in-silico and CRISPR-based migration assays, suggesting that YY1 is a potential therapeutic target for deterring metastasis.

Synthetic autotrophs are a promising platform for sustainable bioproduction using CO 2 as substrate. The methylotrophic yeast Komagataella phaffii has been engineered to use CO 2 as the sole carbon source by integration of the Calvin–Benson–Bassham (CBB) cycle, based on its native methanol assimilating xylulose monophosphate pathway (XuMP) cycle. Initial growth rates were low, but could be doubled by adaptive laboratory evolution (ALE). Beneficial mutations led to a decrease of CBB cycle reactions, indicating further limitations. During this study, temperature was identified as one of the key process parameters to improve autotrophic growth. For this reason, a new round of adaptive laboratory evolution was performed at the identified optimal cultivation temperature of 25°C, resulting in isolates growing up to 50 % faster compared to the control strain. Whole genome resequencing followed by reverse engineering helped to identify first key mutations of the evolved strains. In addition, targeted engineering was performed by increasing the copy number of the key gene of the CBB cycle RuBisCO, which is the bottleneck of carbon fixation. Combining this with the optimal cultivation temperature boosted maximum specific growth rates of the autotrophic K. phaffii strain. In comparison to ALE, the targeted engineering still is lagging behind a bit. Starting from the initial condition, growth was boosted more than 2.5-fold in this study to a maximum of 0.025 h -1 .

ABSTRACT Wnt signaling plays an essential role in organismal development and stem cell maintenance, and is frequently dysregulated in various human diseases. Wnt proteins are lipid-modified, secreted morphogens that activate biological programs in a concentration-dependent manner. Their secretion depends on specialized trafficking components, including the cargo receptor Evi/Wls, and often involves apical re- internalization and transcytosis. However, several critical steps in Wnt/Wg trafficking remain poorly understood. In this study, we conducted an in vivo CRISPR-Cas9 screen in the developing Drosophila wing imaginal disc to uncover novel regulators of Wg secretion. We identified Vps15, a regulatory subunit of the class III phosphatidylinositol 3-kinase (PI3K(III)) complex, as an essential factor for Wg trafficking at the apical membrane. Loss of Vps15 leads to pronounced apical accumulation of Wg in producing cells, elevated extracellular Wg levels, and reduced expression of Wg target genes. Our data indicate that PI3K(III) activity is required for efficient apical endocytosis of Wg, independent of Evi/Wls abundance. These findings reveal a previously unrecognized role for the PI3K(III) and lipid biogenesis in Wg secretion within polarized epithelia, enhancing our mechanistic understanding of Wnt trafficking. Summary statement We identify the PI3K(III) complex as a novel regulator of Wg endocytosis at the apical membrane, revealing a new mechanism controlling Wnt secretion in polarized epithelial tissue.

SUMMARY Animal models of human diseases are an essential component of understanding disease pathogenesis and serve as preclinical models for therapeutic evaluation. Recently human patient genome sequencing has defined unique patient variants that result in disease states with different phenotypes than those observed with null alleles. The UAB Center for Precision Animal Modeling (CPAM) serves to analyze patient variant pathogenicity and disease mechanisms through the generation of animal models. We have optimized a zebrafish gene editing platform to successfully generate 11 patient variants (first round: NF1 R1276Q, NF1 G484R, VMA21 G55V, SPOP D144N, SGO1 K23E, Pex10 H310D, and FKRP C318Y; second round: NF1 R681*, NF1 M992del, P53 R175H, and PKD2 L656W) and 1 research allele ( p53 K120R). We used CRISPR/Cas9 guide directed cleavage along with single-stranded oligodeoxynucleotide (ssODNs) repair templates to generate these models. We evaluated multiple oligo orientations and sizes, but did not find a unified consensus orientation or size that significantly impacted efficiency, emphasizing the need to empirically evaluate multiple variations for the best homology directed repair (HDR) rate. We determined PCR amplicon Next Generation Sequencing (NGS) evaluation of HDR efficiency at the F0 embryo level is best for determining the ideal guide and oligo combination. Further NGS evaluation of DNA from progeny from F0s (germline derived), not F0 biopsy DNA, is essential to identify germline transmitting founders. Surprisingly we find that most founders exhibit a jackpot effect in the germ line but not in the somatic tissue. We found NGS superior to using ICE (Inference of CRISPR Edits) for determining HDR frequency. When applicable, allelic-specific PCR or allelic specific restriction digestion can be used to genotype mutation carrying F1 generation animals, however we demonstrated that false positives occur. Further, we successfully used high resolution melting curve analysis (HRMA) to differentiate and identify F1 animals with patient variants.

Summary The zinc-finger associated domain (ZAD)-containing C2H2 zinc-finger proteins (ZAD-ZnFs) represent the most abundant class of transcription factors that emerged during insect evolution, yet their molecular diversity and biological functions remain largely unclear. Here, we established a systematic CRISPR-based protein-tagging approach that enables direct, unambiguous comparison of nuclear localization and genome-wide binding profiles of endogenous ZAD-ZnFs in developing Drosophila embryos. Evidence is provided that a subset of ZAD-ZnFs forms nuclear condensates through the stacking of the N-terminal ZAD dimerization surface. Disruption of condensation activity leads to misregulation of genome-wide binding profiles and lethality, underscoring its functional and physiological significance in development. Importantly, integrative ChIP-seq and Micro-C data analyses reveal that many ZAD-ZnFs colocalize with core insulator proteins such as CTCF and CP190 to strengthen the formation of topological boundaries. We suggest that the diverse molecular functions of ZAD-ZnFs have evolutionally arisen from their ancestral role as insulator-binding proteins.

Bioplastics represent promising alternatives to petroleum-based plastics, yet their biodegradation remains insufficiently understood. Identifying bacteria capable of degrading bioplastics extracellularly could enhance end-of-life management practices. To investigate Burkholderia's capacities for the degradation of medium-chain-length polyhydroxyalkanoate (mcl-PHA), we screened a panel of Burkholderia strains and identified such capacity in some strains of B. gladioli, B. multivorans, and B. vietnamiensis. To elucidate the genetic basis of this activity, we performed transposon mutagenesis followed by activity-based screening and Tn-seq on B. vietnamiensis LMG 16232. Transposon insertions with negative phenotype were identified in genes encoding a triacylglycerol lipase, a lipase chaperone, type II secretion system components, HTH-type transcriptional activators, and a polyhydroxyalkanoate synthesis repressor. To validate the involvement of these genetic elements in mcl-PHA degradation, we generated insertional mutants using a CRISPR-associated transposase system (CAST). Complete loss or reduced extracellular mcl-PHA depolymerase activity was observed in the CAST mutants, validating their involvement in mcl-PHA degradation. In addition, the interruption of these genetic elements showed a loss of lipase activity, suggesting that the enzymes responsible for mcl-PHA depolymerase are lipases with substrate ambiguity; moreover, docking experiments supported these findings. Together, we identify B. vietnamiensis as a source of enzymes capable of degrading extracellular mcl-PHA and demonstrate the power of combining activity-based screening, Tn-seq, and CAST to rapidly establish gene-to-function links.

Duchenne muscular dystrophy (DMD) is a progressive muscle wasting disease for which there is no cure. There is a critical need for additional therapeutics. Human genome-wide association studies (GWAS) have identified candidate DMD genetic modifiers that could serve as therapeutic targets. Because many GWAS-identified single nucleotide polymorphisms (SNPs) lie in noncoding, putative regulatory regions, it can be challenging to identify which gene(s) are regulated by these SNPs and how gene expression is altered to modify disease severity even with extensive in silico modeling. We analyzed expression of zebrafish orthologs of putative DMD modifiers and showed almost all are comparably expressed in wild-type and dmd mutant zebrafish at three different stages of disease. To model decreased expression of candidate modifiers, we pursued a zebrafish CRISPR-based screening approach, which we validated by testing zebrafish orthologs of two extensively studied DMD modifiers, LTBP4 and THBS1. We then tested candidates from the most recent GWAS and demonstrate that galntl6, man1a1, etaa1a;etaa1b, and adamts17 are bona fide DMD modifiers. Our findings demonstrate the utility of zebrafish for DMD genetic modifier screening and characterizing modifier function.

CRISPR base editors enable scalable targeted DNA mutagenesis and are a powerful tool for the interpretation of gene function, the study of variants of unknown significance, and disease modelling. Existing guide RNA (gRNA) design tools focus on engineering a small number of targeted variants in a given gene or lack comprehensive functional annotation of target sequences. Here we developed BEstimate, a flexible computational pipeline that systematically specifies base editor gRNA target sites, generates gRNA off-target predictions, and rich functional, structural and clinical annotations of targeted edits. BEstimate accommodates custom gRNA design against variant alleles, improving editing accuracy and facilitating the programmed reversion of disease variants. BEstimate is a freely available, versatile tool for designing gRNA libraries and analysing base editor screens.

Computational models have become essential tools for understanding signalling networks and their non-linear dynamics. However, these models are typically constructed manually using prior knowledge and can be over-reliant on study bias. These limitations hinder their ability to make accurate predictions and incorporate new evidence. Scaling up the construction of models to take advantage of increasingly abundant ‘omics data can bridge these gaps by providing a comprehensive view of signalling events and how they influence cellular phenotypes. In this study, we present MAGELLAN, a method leveraging message passing graph neural networks to build computational models directly from pathway data and discrete rules representing experimental results. We used this to construct a computational model of breast cancer signalling and re-parameterize a previously published non-small cell lung cancer (NSCLC) model, showing that MAGELLAN can predict genetic dependencies and achieve comparable model quality to expert-curated and manually trained models. Our approach enables the integration of prior knowledge networks and experimental data to build predictive models that are mechanistically interpretable. This approach simplifies model creation, making it more accessible and practical for experimentalists, and supports broader applications in drug discovery and biological research.

Abstract The developmentally dynamic stem cell-like subpopulation in cancers known as cancer stem cells (CSCs) metastasize efficiently, form new tumours, and are resistant to current therapeutics. Understanding the molecular mechanisms of the stem cell-like state could help eliminate CSCs via novel therapeutic applications in aggressive cancers such as pancreatic ductal adenocarcinoma (PDAC). Here, we performed a compound screen uncovering that inhibition of Hypoxia-inducible factor prolyl hydroxylase 2 (PHD2) supports CSCs, whereas a novel proline-uncompetitive glutamyl-prolyl-tRNA synthetase (GluProRS) inhibitor NCP26 eliminates CSCs. By using a multiomic strategy with transcriptomics and quantitative secretomic analyses, we uncovered that TGFβ/Activin signalling induces HIF1A-mediated pseudohypoxia via ɑ-Ketoglutarate depletion due to P4HA1 intracellular upregulation. In turn, HIF1A and SMAD2/3 form a transcriptional complex that regulates CSC self-renewal, invasiveness and chemoresistance. NCP26 has several anti-tumour effects, including direct activation of the Integrated Stress Response (ISR) and indirect inhibition of the pseudo-hypoxia circuitry mediated by SMAD2/3-HIF1A that deposits collagen in ECM. Furthermore, GluProRS is secreted from PDAC cells and elevated in PDAC patient blood compared to healthy and pancreatitis patients. Collectively, the secreted GluProRS protein could be a useful biomarker for early PDAC detection, and its inhibitor NCP26 is an attractive candidate for combined chemotherapeutic applications.

Abstract Background BRAT1 (BRCA1-associated ataxia telangiectasia mutated activator 1) plays a crucial role in several vital biological processes, including the DNA damage response and the maintenance of mitochondrial homeostasis. Dysfunction in BRAT1 leads to a range of clinical phenotypes, with the majority of affected individuals succumbing before reaching one year of age. Results Through an analysis of previous literature, the homozygous BRAT1 p.V62E mutation (GTG to GAG) was selected to construct a mouse model. Homozygous BRAT1 p.V62E knock-in mice with a C57BL/6J background were generated using CRISPR/Cas9 technology, and the point mutation was confirmed by Sanger sequencing. The results revealed no significant differences between the mutant mice and wild-type controls during low-intensity testing. However, the mutant mice exhibited enhanced endurance during high-intensity testing. RNA sequencing analysis identified ten differentially expressed genes in the gastrocnemius muscle and four differentially expressed genes in the brain tissue of the mutant mice. Conclusions This represents the first successful construction of a BRAT1 mutant mouse model. This achievement not only provides confidence for developing additional mouse models but also offers a valuable perspective for understanding the relationship between BRAT1 and mitochondrial function. The mouse model demonstrates a degree of consistency with the clinical phenotypes observed in patients and may serve as a predictive tool for patient prognosis.

ABSTRACT Phosphorylation, a crucial post-translational modification (PTM), plays a central role in cellular signaling and disease mechanisms. Mass spectrometry-based phosphoproteomics is widely used for system-wide characterization of phosphorylation events. However, traditional methods struggle with accurate phosphorylated site localization, complex search spaces, and detecting sequences outside the reference database. Advances in de novo peptide sequencing offer opportunities to address these limitations, but have yet to become integrated and adapted for phosphoproteomics datasets. Here, we present InstaNovo-P, a phosphorylation specific version of our transformer-based InstaNovo model, fine-tuned on extensive phosphoproteomics datasets. InstaNovo-P significantly surpasses existing methods in phosphorylated peptide detection and phosphorylated site localization accuracy across multiple datasets, including complex experimental scenarios. Our model robustly identifies peptides with single and multiple phosphorylated sites, effectively localizing phosphorylation events on serine, threonine, and tyrosine residues. We experimentally validate our model predictions by studying FGFR2 signaling, further demonstrating that InstaNovo-P uncovers phosphorylated sites previously missed by traditional database searches. These predictions align with critical biological processes, confirming the model’s capacity to yield valuable biological insights. InstaNovo-P adds value to phosphoproteomics experiments by effectively identifying biologically relevant phosphorylation events without prior information, providing a powerful analytical tool for the dissection of signaling pathways.

Classification of high-dimensional information is a ubiquitous computing paradigm across diverse biological systems, including organs such as the brain, down to signaling between individual cells. Inspired by the success of artificial neural networks in machine learning, the idea of engineering genetic circuits that operate as neural networks emerges as a strategy to expand the classification capabilities of living systems. In this work, we design these biomolecular neural networks (BNNs) based on the molecular sequestration reaction, and experimentally characterize their behavior as linear classifiers for increasing levels of complexity. Initially, we demonstrate that a static, DNA-based system can effectively prototype a linear classifier, though we also identify its limitations to easily tune the slope of the decision boundary it generates. We then propose and experimentally validate a BNN at the protein level using a cell-free transcription–translation (TXTL) system, which overcomes the DNA-based system’s limitation and behaves as a linear classifier even before it reaches its steady state (or, equivalently, out-of-equilibrium). Ultimately, we test a CRISPR-based design and its out-of-equilibrium behavior in a biological context by successfully constructing a linear classifier within mammalian cells. Overall, by leveraging mathematical modeling and experimental automation, we establish molecular sequestration as a universal scheme for implementing neural networks within living systems, paving the way for transformative advances in synthetic biology and programmable biocomputing systems.

Lipid nanoparticles (LNPs) have emerged as a transformative platform for mRNA delivery, enabling vaccines and gene editing with transient expression and high cargo capacity. However, their potential for ocular gene editing remains underexplored. In this study, we assessed the transduction efficiency, inflammatory response, and gene editing capability of LNP-encapsulated mRNA in murine eyes. Intravitreal delivery of LNPs achieved targeted mRNA expression in the trabecular meshwork (TM) with superior specificity and efficiency compared to adenoviral or adeno-associated viral vectors, while inducing minimal microglial activation in the retina. Using LNPs co-encapsulating SpCas9 mRNA and sgRNA, we demonstrated efficient CRISPR-mediated knockout (KO) of Matrix Gla Protein (Mgp), a key inhibitor of TM calcification. Mgp-KO eyes exhibited sustained intraocular pressure (IOP) elevation and anterior chamber deepening with normal anterior chamber angle, recapitulating key features of primary open-angle glaucoma (POAG). Chronic IOP elevation led to reactive Müller gliosis and ganglion cell complex thinning, reflecting retinal stress and progressive neurodegeneration. Our findings establish LNP-CRISPR as a safe and efficient system for TM-targeted gene editing, with broad applicability in glaucoma pathogenesis modelling and therapeutic discovery.

ABSTRACT Retinitis Pigmentosa (RP) is an inherited retinal dystrophy characterized by the progressive loss of rod photoreceptors. Sector RP is a form of RP, where degeneration originates in the inferior retina, mainly influenced by light exposure. Over 200 RHO variants are pathogenic and associated with autosomal dominant RP. RHO M39R is one of the most common RHO variants linked to sector RP in the UK. A knock-in (KI) mouse model expressing Rho M39R was generated and characterized to investigate the mechanisms of degeneration associated with this variant and explore novel therapeutic strategies for rhodopsin sector RP. Under ambient light, Rho M39R/+ KI mice exhibited impaired retinal function by ERG, with some defects in OS ultrastructure, but retained normal outer nuclear layer (ONL) thickness. Repeated exposure to bright light led to photoreceptor loss. In contrast, Rho M39R/M39R KI mice in ambient light displayed severe retinal dysfunction, ONL thinning, and grossly abnormal OS ultra structure. In homozygous mice, a single bright light exposure significantly reduced ONL thickness within 48 h. The rescue of these models was achieved through reduced light exposure and pharmacological intervention. Rearing in dim red light (red cage condition) restored ERG responses in Rho M39R/+ KI mice and improved ONL thickness in Rho M39R/M39R KI mice. Transcriptomic analysis in Rho M39R/M39R KI mice revealed upregulation of Sphingosine 1-P Receptor (S1PR) transcripts. Treatment with the S1PR agonist Fingolimod (FTY720) before bright light exposure significantly reduced degeneration, demonstrating a protective effect in both heterozygous and homozygous models and suggesting potential a therapeutic approach for sector RP patients.

Delayed diagnosis of Mendelian disease substantially prevents early therapeutic intervention that could improve symptoms and prognosis. One major contributing challenge is the functional interpretation of non-coding variants that cause disease by altering splicing and/or gene expression. We identified two siblings with glycogen storage disease (GSD) type IX γ2, both of whom had a classic clinical presentation, enzyme deficiency, and a known pathogenic splice acceptor variant on one allele of PHKG2 . Despite the autosomal recessive nature of the disease, no variant on the second allele was identified by gene panel sequencing. To identify a potential missing second pathogenic variant, we completed whole genome sequencing (WGS) and detected putative deep intronic splicing variant in PHKG2 in both siblings. We confirmed the functional splicing effects of this variant using short-read and long-read RNA-seq on patient blood and a HEK293T cell model in which we installed the variant using CRISPR editing. Using the cell model, we demonstrated multiple biochemical and cellular impacts that are consistent with GSD IX γ2, and a reversal of aberrant splicing using antisense splice-switching oligonucleotides. In doing so, we demonstrate a novel and robust pathway for detecting, validating, and reversing the impacts of novel non-coding causes of rare disease.

The United Kingdom (UK) is home to many local and rare livestock breeds. The local breed populations are highly adapted to specific environments in the UK and these and other rare breeds provide solutions to niche needs. Rare breeds often contribute more than expected to overall species genetic diversity, which is important because genetic variation is needed for adapting to new challenges. It is therefore very important from both UK and global perspectives to maintain genetic diversity of rare livestock breeds in the UK, and to do this, we need to evaluate the monitoring of genetic diversity to identify gaps in our knowledge and prioritise resources reserved for conservation purposes. The objectives of this study were to survey the literature to: (1) summarise genetic/genomic characterisation (effective population size (N e ) and inbreeding) of domestic populations (livestock and equine) in the UK and Ireland; (2) compare number of populations on the UK’s Rare Breed Survival Trust (RBST) watch list and the number of UK and Irish populations in the peer-reviewed literature with inbreeding, genetic diversity and/or N e estimates; and (3) compare annually reported (census-based) estimates of N e with (inbreeding and DNA-based) estimates from peer-reviewed literature. We found a total of 37 publications with N e or inbreeding estimates for UK or Ireland livestock populations, published from 1975 to 2024. While many (42%) of the breeds on the RBST watchlist have been included in publications, there are still many breeds, and a few species (turkey, duck and geese) with no publicly available pedigree- or DNA-based genetic diversity measures. We found census-based N e estimates were, on average, higher than DNA-based estimates, likely due to violated assumptions when estimating N e with census-based data because of the way livestock mating systems are designed. Most peer-reviewed papers estimated genetic diversity measures using pedigree, microsatellite markers, or SNP markers. To identify breed-unique variants responsible for adaptive traits in rare breeds, more studies using whole-genome sequencing will be needed. Altogether, we have summarised the genetic diversity estimates on UK livestock populations, identifying gaps in knowledge.

Pseudomonas aeruginosa is a major pathogen associated with hospital-acquired infections, and the spread of carbapenem-resistant isolates highlights the urgency of developing non-conventional therapies, such as phage therapy. For this alternative to be effective, un-derstanding phage-host interactions is crucial for the selection of candidate phages and offers new insights into these dynamics. Background/Objectives: This study aimed to characterize prophage diversity in clinical P. aeruginosa genomes, assess the relationship between phages and the CRISPR/Cas system, and investigate the potential role of phages in disseminating resistance genes. Methods: A total of 141 genomes from Brazilian hos-pitals were analyzed. Prophage detection was performed using VIBRANT, and in silico analyses were conducted to evaluate taxonomic diversity, presence of resistance genes, phage life cycle, genomic distribution, and the presence of the CRISPR/Cas system. Re-sults: In total, 841 viral sequences were identified, with a predominance of the class Cau-doviricetes and high overall phage diversity. No statistically significant difference was ob-served in the number of prophages between isolates with and without CRISPR/Cas sys-tems. Phages carrying resistance genes were detected in isolates harboring the type I-C CRISPR/Cas system. Additionally, prophages showed no preference for specific insertion sites along the bacterial genome. Conclusions: These findings provide evidence of a well-established phage-host relationship. The dual role of phages—as vectors of antimi-crobial resistance and as potential therapeutic agents—reflects their dynamic impact on bacterial communities and reinforces their importance in developing new strategies to combat antimicrobial resistance.

Invasive species inflict major ecological, economic, social, and cultural harm worldwide, highlighting the urgent need for innovative and effective control strategies. Genome editing offers exciting possibilities for creating highly targeted control methods for invasive species. Here, we demonstrate CRISPR-Cas9 genome editing in the cane toad ( Rhinella marina ), one of Australia’s most notorious invasive species, by targeting the tyrosinase gene to produce albino phenotypes that provide clear visual markers for assessing editing efficiency. Microinjection of Cas9 protein and guide RNAs into one-cell zygotes resulted in 87.6% of mosaic larvae displaying nearly complete albinism, with 2.3% exhibiting complete albinism. For completely albino individuals, genomic analysis confirmed predominantly frameshift mutations or large deletions at the target site, with no wild-type alleles detected. Germline transmission rates reflected the extent of albinism in the mosaic adult, where we achieved maternal germline transmission rates of almost 100%. This technology, representing the first application of CRISPR-Cas9 in the Bufonidae family, opens possibilities for exploring both basic research questions and strategies for population control.

ABSTRACT Cellular resource limitations create unintended interactions among synthetic gene circuit modules, compromising circuit modularity. This challenge is particularly pronounced in circuits with positive feedback, where uneven resource allocation can lead to Winner-Takes-All (WTA) behavior, favoring one module at the expense of others. In this study, we experimentally implemented a Negatively Competitive Regulatory (NCR) controller using CRISPR interference (CRISPRi) and evaluated its effectiveness in mitigating WTA behavior in two gene circuits: dual self-activation and cascading bistable switch. We chromosomally integrated a tunable dCas9 gene and designed module-specific gRNAs, with each module encoding its own gRNA to self-repress via competition for limited dCas9. This configuration introduces strong negative feedback to the more active module while reallocating resources to the less active one, promoting balanced module activation. Compared to the control group lacking dCas9-mediated repression, the NCR controller significantly increased module coactivation and suppressed WTA behavior. Our quantitative results demonstrate that NCR provides an effective strategy for regulating resource competition and improving the modularity of synthetic gene circuits.

Zoonotic viruses such as hantaviruses and influenza A viruses present a threat to humans and livestock. There is thus a need for methods that are rapid, sensitive, and relatively cheap to detect infections with these pathogens early. Here we use an amplification-free CRISPR-Cas13-based assay, which is simple, cheap and field-deployable, to detect the presence or absence of genomic hantavirus or influenza A virus RNA. In addition, we evaluate whether the use of multiple CRISPR RNAs (crRNAs) can improve the sensitivity of this amplification-free method. We demonstrate that for the hantaviruses Tula Virus (TULV) and Andes Virus (ANDV) a combination of two or three crRNAs provides the best sensitivity for detecting viral RNA, whereas for influenza virus RNA detection, additional crRNAs provide no benefit. We also show that the amplification-free method can be used to detect TULV and ANDV RNA in tissue culture infection samples and influenza A virus RNA in clinical nasopharyngeal swabs. In clinical samples, the Cas13 assay has an 85% agreement with RT-qPCR for identifying a positive sample. Overall, these findings indicate that amplification-free CRISPR-Cas13 detection of viral RNA has potential as a tool for rapidly detecting zoonotic virus infections.

Abstract Generating mammalian gametes with a skewed sex ratio has thus far eluded empirical confirmation. The utilization of such genetically engineered organisms would offer the potential to curtail the necessity for culling animals of undesirable sex, mitigate resource wastage, and alleviate superfluous labor burdens. In this study, we introduce a transgenic male mouse lineage, which consistently yields predominantly female progeny (comprising up to ~90% of the total offspring). This accomplishment was made possible by integrating a controllable genetic cassette onto the Y chromosome. The cassette encodes dCas9 and RNA guides that selectively silence a spermatid maturation gene. After the separation of X and Y gametes during meiosis, gametes containing an X chromosome develop normally, while those harboring the engineered Y chromosome, subjected to dCas9 silencing of the spermatid maturation gene, do not mature properly. Indeed, some spermatozoa from the transgenic mice exhibit a unique morphology, associated with the absence of the maturation gene. Notably, the resultant female offspring do not inherit the genetically engineered Y chromosome and are thus not genetically modified. Importantly, the litter size of the transgenic mice remains unchanged compared to the wild type. These findings represent the potential of genetic engineering to yield sex-biased litters of full size without compromising genetic integrity, marking a pioneering advancement in this field of study.