Co-editing strategies have emerged as an approach to facilitate the selection of CRISPR/Cas-mediated mutants in a transgene-free manner: the gene of interest is edited together with a reporter gene, whose mutation can be selected visually or pharmacologically. In this work, we asses the impact of editing the well-used reporter Acetolactate synthase (ALS) on plant development and metabolome. We show that the desired mutation in ALS1 (P186S) conferring selectable herbicide resistance trait does not show significant impact on the plant morphology and physiology but that the additional mutations resulting from the same sgRNA can result in reduced vegetative vigor and altered metabolomic profiles in tomato.

Background Polycomb Repressive Complex 2 (PRC2) modulates chromatin accessibility and architecture to direct tissue-specific gene expression. PRC2 function is frequently altered in cancer by loss-of-function mutation or deletion, but the downstream effects on transcriptional regulation are incompletely understood. Results To gain insights into these mechanisms, we performed a holistic analysis of epigenomic and transcriptional changes in an isogenic model of acute myeloid leukemia (AML) with heterozygous EZH2 deletion that mimics reduced PRC2 function in patient leukemias. PRC2-depleted cells had diverse gene expression changes, including a bias towards more immature monocyte-lineage transcriptional signatures. PRC2 depletion also correlated with marked increases in chromatin accessibility genome-wide, with 10-45% increases in ATAC-seq peaks in EZH2+/− clones. These changes were accompanied by decreased H3K27me3 and increased H3K27ac levels in CUT+RUN assays that were incompletely linked to transcriptional activity. Despite these generalised changes, 3D chromatin architecture assessed by Hi-C was largely maintained, with H3K27me3 preferentially lost in regions with low DNA-DNA contact frequency. Surprisingly, some regions gained broad H3K27me3 domains at heavily compacted chromatin. We notably saw compartmentalisation changes upstream of the transcriptionally upregulated fetal hematopoiesis gene LIN28B in EZH2+/− cells, with corresponding activation of a LIN28B-specific transcriptional program, including upregulation of the CDK6 oncogene. These results correlated with EZH2+/− cell phenotype, including decreased cellular proliferation and increased resistance to CDK6 inhibitor palbociclib. Conclusions Our findings suggest that PRC2 depletion pleiotropically affects AML transcriptional regulation to directly impact cell phenotype and treatment responsiveness, which may partially explain the aggressive biology seen in these cases.

Cell signaling plays a critical role in regulating cellular state, yet uncovering regulators of signaling pathways and understanding their molecular consequences remains challenging. Here, we present an iterative experimental and computational framework to identify and characterize regulators of signaling proteins, using the mTOR marker phosphorylated RPS6 (pRPS6) as a case study. We present a customized workflow that uses the 10x Flex assay to jointly profile intracellular protein levels, transcriptomes, and CRISPR perturbations in single cells. We use this to generate a “glossary” dataset of paired protein–RNA measurements across targeted perturbations, which we leverage to train a predictive model of pRPS6 levels based solely on transcriptomic data. Applying this model to a genome-wide Perturb-seq dataset enables in silico screening for pRPS6 and nominates novel regulators of mTOR signaling. Experimental validation confirms these predictions and reveals mechanistic diversity among hits, including changes in signaling output driven by anabolic activity, cellular proliferation and multiple stress pathways. Our work demonstrates how integrated experimental and computational approaches provide a scalable framework for multimodal phenotyping and discovery.

Anti-bacteriophage systems like restriction-modification and CRISPR-Cas have DNA substrate specificity mechanisms that enable identification of invaders. How Gabija, a highly prevalent nuclease-helicase anti-phage system, executes self- vs. non-self-discrimination remains unknown. Here, we propose that phage-encoded DNA end-binding proteins that antagonize host RecBCD sensitize phages to Gabija. When targeting temperate phage D3 in Pseudomonas aeruginosa, Gabija functions early by preventing phage genome circularization in a non-abortive manner. Phage and plasmid DNA-end sensitivity to Gabija is licensed by a phage exonuclease and ssDNA-annealing protein. Unrelated F8 and JBD30 phages are sensitized to Gabija by Gam_Mu, a distinct DNA end-binding protein that antagonizes loading of the host repair complex RecBCD. Escape phages lacking these end-binding proteins become protected from Gabija by RecBCD activities, which also prevent Gabija from targeting self-DNA. Therefore, we propose that Gabija antagonizes circularization of linear DNA devoid of RecBCD as a mechanism to identify foreign invaders.

ABSTRACT Synthetic biology enables the integration of sophisticated genetic programs into microorganisms, transforming them into potent vehicles for therapeutic applications. Engineering strategies for microorganisms are rapidly evolving, offering promising solutions for cancer therapy, microbiome modulation, digestive health support, and beyond. Developing novel tools to engineer safe, nonpathogenic microbial platforms is essential for advancing clinical therapies. In this work, we present an innovative engineering approach for the probiotic Escherichia coli Nissle (EcN), aimed at creating a safe and efficient chassis for the bioproduction of therapeutics. The EcN endogenous pM1 and pM2 plasmids were cured and re-engineered to introduce a CRISPR-Cas12 chromosome shredding device and a therapeutic-producing genetic circuit, thereby generating a nonproliferative therapeutic-delivery system. Next, we build an AI-based bioinformatic pipeline to predict Anticancer-Cell-Penetrating Peptides (ACCPP) candidates. As a proof-of-concept, a selected ACCPP was produced in the engineered EcN chromosome-shredded (CS) chassis. This strategy yields a robust and controllable platform for the safe production and delivery of therapeutics, paving the way for the future development of microbial therapies and their clinical applications. GRAPHICAL ABSTRACT

Mutations in the ciliary protein INPP5E, encoded by inositol polyphosphate-5-phosphatase E, can cause retinal degeneration as part of the ciliopathy Joubert Syndrome or non-syndromic retinitis pigmentosa (RP). INPP5E regulates the membrane makeup of the primary cilium, however its function in the specialized sensory photoreceptor cells of the human retina remain unclear. Here we utilize control and CRISPR/Cas9-generated INPP5E knock-out ( INPP5E KD ) human induced pluripotent stem cells (iPSCs) to generate retinal organoids (ROs). Through proteomic and immunofluorescence analysis we show that INPP5E plays an important role in early retinal development and photoreceptor progenitor cell differentiation. In mature ROs, INPP5E localizes to the connecting cilium of photoreceptors, and the loss of INPP5E leads to altered localization of ARL13B and Rhodopsin in mature photoreceptors. Furthermore, photoreceptor outer segment structure is affected, leading to elongated outer segment membranes in both cone and rod photoreceptors, suggesting an important role for INPP5E in photoreceptor outer segment membrane biogenesis. Together, these data underline the importance of INPP5E in retina development and photoreceptor structure and highlight the usability of retinal organoids to study protein function in a human context.

Cancer treatment remains challenging due to heterogeneous responses to immunotherapy across patients and tumor types. Innovative strategies are required to overcome immune evasion. We have identified the splicing factor SLU7 as essential for the survival of cancer cells from diverse origins. SLU7 knockdown induces R-loop accumulation, transcription-dependent genomic instability, DNA damage, and replication catastrophe, together with aberrant splicing and inhibition of nonsense-mediated mRNA decay (NMD) and/or DNA methylation. These alterations lead to the expression of neoantigens, interferon B1, endogenous retroviruses, and cancer-testis antigens, which would enhance tumor immunogenicity. Therefore, we propose SLU7 targeting as a dual-action therapy, combining direct tumor suppression with immune activation. Using various murine cancer models, including orthotopic liver tumors, and multiple molecular strategies—such as inducible CRISPR/Cas9, systemic delivery of chimeric siSLU7–nucleolin aptamers (APTASLU), and intratumoral injection of siSLU7-loaded nanoparticles—we show that distinct siSLU7 sequences and delivery platforms effectively inhibit tumor growth. Furthermore, SLU7 silencing synergizes with immune checkpoint inhibitors, amplifying anti-tumor responses. Our in vivo data demonstrate that SLU7 is a promising, versatile target for diverse cancers. Its multimodal mechanism offers potential to overcome tumor heterogeneity, reverse immune tolerance, and enhance immunotherapy efficacy.

Background: Collagen fibrils are the primary supporting scaffolds of vertebrate tissues, but the mechanism of assembly is unclear. Methods Here, using CRISPR-tagging of type I collagen, high-resolution light imaging, and SILAC labelling, we elucidated the cellular mechanism underlying the spatiotemporal assembly of collagen fibrils in cultured fibroblasts. Results Our findings reveal the multifaceted trafficking of collagen, including constitutive secretion, intracellular pooling, and plasma membrane-directed fibrillogenesis. Notably, we differentiated the processes of collagen secretion and fibril assembly and identified the crucial involvement of endocytosis in the regulation of fibril formation. By employing Col1a1 knockout fibroblasts, we demonstrated the incorporation of exogenous collagen into the nucleation sites at the plasma membrane through these recycling mechanisms. Conclusions Our study sheds light on a complex and previously unidentified collagen assembly process and its regulation of health and disease. Mass spectrometry data were available via ProteomeXchange with the identifier PXD036794.

How genomic changes translate into organismal novelties is often confounded by the multi-layered nature of genome architecture and the long evolutionary timescales over which molecular changes accumulate. Coleoid cephalopods (squid, cuttlefish, and octopus) provide a unique system to study these processes due to a large-scale chromosomal rearrangement in the coleoid ancestor that resulted in highly modified karyotypes, followed by lineage-specific fusions, translocations, and repeat expansions. How these events have shaped gene regulatory patterns underlying the evolution of coleoid innovations, including their large and elaborately structured nervous systems, novel organs, and complex behaviours, remains poorly understood. To address this, we integrate Micro-C, RNA-seq, and ATAC-seq across multiple coleoid species, developmental stages, and tissues. We find that while topological compartments are broadly conserved, hundreds of chromatin loops are species- and context-specific, with distinct regulation signatures and dynamic expression profiles. CRISPR-Cas9 knockout of a putative regulatory sequence within a conserved region demonstrates the role of loops in neural development and the prevalence of long-range, inter-compartmental interactions. We propose that differential evolutionary constraints across the coleoid 3D genome allow macroevolutionary processes to shape genome topology in distinct ways, facilitating the emergence of novel regulatory entanglements and ultimately contributing to the evolution and maintenance of complex traits in coleoids.

Streptococcus suis is a major pig pathogen with zoonotic potential, posing an occupational risk to farmers and meat handlers. We characterised 110 S. suis strains from diseased pigs in Ireland (2005–2022) using whole-genome sequencing to investigate population structure and phage-host dynamics. We identified fifteen distinct serotypes, with serotypes 9 and 2 being the most dominant. In silico multi-locus sequence typing revealed high diversity within the collection, identifying several sequence types (STs), including 26 novel STs. Investigation of strain-level genomic clustering using PopPUNK against global S. suis genomes showed that the Irish isolates were phylogenetically dispersed across the broader global S. suis population rather than clustering in a single clonal group. The majority of Irish isolates fall within the ten established pathogenic lineages, including the highly virulent zoonotic lineage 1. A stable endemic clonal lineage was identified among Irish isolates, showing minimal genetic variation over a decade. Prophage analysis revealed novel viral taxa that were interspersed among known streptococcal phages, rather than clustering distinctly. Restriction-modification systems were the predominant anti-viral defence systems identified across genomes. CRISPR-Cas systems were present in limited strains but showed substantial targeting bias toward full-length prophages, indicating ongoing phage pressure. CRISPR spacers matched non- S. suis streptococcal phages, and phylogenomic analysis revealed that Vansinderenvirus phages clustered with S. suis rather than other S. thermophilus phages, suggesting evolutionary connections between phage lineages infecting different streptococci. This study presents the first comprehensive genomic characterisation of S. suis in Ireland, revealing a diverse population with significant implications for animal and human health.

Although haemoglobin variants are prevalent in low- and middle-income countries, the exact disease burden remains unknown due to a lack of diagnostic capacity. Traditionally, routine clinical haemoglobin variant diagnostics have relied on electrophoresis, which separates the haemoglobins based on size and charge differences. However, electrophoresis-based assays are limited in depth and coverage due to their inability to separate co-migrating variants at the pH employed. Importantly, for genetic counselling of pre-marital couples and prenatal screening of inherited haemoglobinopathy risk, molecular-based assays are required. Here, we leveraged the specificity and dual mismatch intolerance of en31FnCas9 to achieve differential identification of haemoglobin variants S, C, D, and E. Moreover, we demonstrate reliable differential detection of homozygous, heterozygous and compound states of haemoglobin variants due to en31FnCas9s intolerance to dual mismatch in the respective gRNA-target DNA complementarity. Furthermore, we coupled our en31FnCas9-based haemoglobin variant detection to the signal enhancement of recombinase polymerase amplification (RPA) to achieve reliable differential detection of haemoglobin variants from non-invasive saliva and urine samples in <60 minutes. Taken together, our study demonstrates the feasibility of functionalization of CRISPR-based diagnostics as a point-of-care technique towards achieving the democratisation of haemoglobin variant diagnosis in resource-limited settings.

Plasmodium species malaria parasites require invasion and replication within red blood cells to cause disease. Merozoite surface proteins (MSPs) are proposed to play a role in attachment of merozoites to RBCs and have long been considered as potential vaccine targets, but their functions during invasion are largely unknown. We applied targeted gene editing to investigate MSP4 and 5 function in P. falciparum , which causes most malaria mortality, and P. knowlesi , an in vitro culturable zoonotic species closely related to the widespread P. vivax . CRISPR-Cas9 gene-editing revealed that P. knowlesi MSP4 was not required for parasite growth in vitro . While P. knowlesi MSP5 could be functionally replaced by P. vivax MSP5, it was refractory to gene deletion. We confirmed the opposite for two different P. falciparum laboratory isolates where MSP4 is essential but MSP5 is dispensable. Attempts to select for reliance on the non-essential MSP (e.g. P. knowlesi MSP4 or P. falciparum MSP5) through long-term growth of inducible knock-out parasites, or via chimeric complementation of the essential MSP4 or 5 with the essential MSP from the other species, were unsuccessful. Live cell filming revealed a severe cell-entry defect with conditional knock-down of MSP5 protein expression in P. knowlesi . This study demonstrates differential importance of MSP4 and MSP5 during merozoite RBC invasion across human infecting malaria species, emphasises that vaccine candidates must be considered individually for the two most prominent human malarias and promotes MSP5 as a potential vaccine candidate for P. knowlesi and P. vivax . Significance For a malaria parasite to cause disease, the merozoite form of the lifecycle has to infect and replicate within human red blood cells. Proteins on the surface of the merozoite are considered as promising vaccine candidates, but the functions of these proteins are poorly understood. Here we demonstrate that two structurally similar merozoite surface proteins (MSP), MSP4 and MSP5, have differential importance between one human infecting malaria species compared to a second. The finding that MSP4 is essential for growth in one species, and MSP5 in the other, has implications for understanding invasion biology of malaria parasites and highlights that even structurally similar vaccine targets may need to be chosen specifically for each human infecting malaria species.

Succinate dehydrogenase (SDH)-deficient paraganglioma and pheochromocytoma (PPGL) are rare neuroendocrine tumors for which no effective targeted therapies currently exist. To uncover new potential therapeutic targets, we performed an unbiased CRISPR-Cas9 genetic screen in immortalized mouse chromaffin cells (imCCs) with and without Sdhb loss. Our screen identified genes that differentially affect cell proliferation in Sdhb -deficient versus normal imCCs. Notably, several subunits of the transcriptional Mediator complex emerged as potential tumor suppressors, as their loss selectively promoted growth of Sdhb -deficient cells. Most strikingly , we found that the neddylation pathway—required for ubiquitin-mediated selective protein degradation—plays a critical role in controlling cell growth and survival in Sdhb -deficient imCCs. Specifically, loss of the neddylation regulator Ube2m led to increased proliferation, while loss of Ube2f suppressed growth of Sdhb -deficient imCCs. Consequently, global neddylation inhibitor MLN4924 (Pevonedistat) and UBE2F-CRL5 axis inhibitor HA-9104 were shown to downregulate neddylation, suppressing UBE2F activity and selectively inhibiting growth of Sdhb -deficient imCCs. This unexpected result highlights the neddylation pathway as a promising druggable vulnerability in this cell culture model of SDH-deficient PPGL.

Efficient gene integration using RNA-guided endonucleases has not yet been achieved in the mitochondrial genome. Import of nucleic acids into mitochondria, a controversial feature, is essential for implementation of Cas9-mediated genome engineering of mitochondria. Import of short RNAs naturally occurs in mitochondria, and several putative import mechanisms and determinants have been proposed. However to date, import of gene-length RNA, required for gene integration in the mitochondrial genome, has never been described. The goal of this study was to devise and test experimental strategies to detect and improve the import of mRNA-sized RNA in mitochondria, using S. cerevisiae as model. A first fluorescence-based screening approach, relying on mitochondrial import of a fluorescent protein encoding mRNA was analyzed by fluorescence measurements, western blot and mRNA-FISH. Confounding results obtained with these different techniques made it difficult to unambiguously conclude on the occurrence of import of mRNA-sized RNAs into mitochondria. An adaptive laboratory evolution (ALE) approach, imposing a strong selection pressure for mRNA import to mitochondria, was then designed and tested to improve mitochondrial mRNA import. While the ALE approach did not improve mitochondrial mRNA import in the present study, it is a promising, unambiguous method for future studies testing different RNAs or mutants. The present study highlights remaining challenges in analytical techniques to identify RNA import to mitochondria, and introduces a novel application of ALE for studies on mitochondrial import of short and long RNA species.

Abstract Nesfatin-1 is a brain-gut peptide encoded by the nucleobindin-2 (NUCB2) gene. We previously demonstrated that a reduced level of nesfatin-1 in the cerebrospinal fluid, induced by intracerebroventricular injection of a nesfatin-1 antibody, is associated with degeneration of the nigrostriatal dopaminergic system. In combination with evidence that nesfatin-1 mediated the rescue of toxicant induced dopaminergic (DAergic) neuron loss in the substantia nigra (SN), as well as reduced nesfatin-1 levels in the blood of patients with Parkinson’s disease (PD), we raise the hypothesis that nesfatin-1 may be essential for the survival of DAergic neurons in SN in mice. In the present study, we found that whole-body Nucb2 knockout via CRISPR/Cas9 technology in mice led to nigrostriatal dopaminergic system degeneration, as evidenced by a reduction in tyrosine hydrolyses-immunoreactivity neurons in the SN, decreased levels of dopamine and its metabolites in the striatum, and mitochondrial and nuclear impairment in the SN. The underlying mechanism may involve oxidative stress and neuroinflammation induced by down-regulation of circadian rhythm-related gene expression. Furthermore, Nucb2 deletion in mice leads to intestinal microecological imbalance, disorder of the bacterial community structure, metabolic homeostasis disruption, and decreased abundance of some sleep rhythm-related bacterial communities and metabolites. Our findings reported that nesfatin-1 plays a role in maintaining the normal function of the nigrostriatal dopaminergic system, which may provide new therapeutic targets for PD.

Abstract Background Over 300 mutations in PSEN1 have been identified as causes of early-onset Alzheimer’s disease (EOAD). While these include missense mutations and a few insertions, deletions, or duplications, none result in open reading frame shifts, and all alter γ-secretase function to increase the long/short Aβ ratio. Methods We identified a novel heterozygous PSEN1 nonsense variant, c.325A > T, in a patient and his father, both presenting with EOAD, resulting in the substitution of lysine 109 with a premature stop codon at position (p.K109*). This produces a truncated 109 amino acid (aa) N-terminal PSEN1 fragment. Functional characterization was performed using overexpression models and a heterozygous mouse model (Psen1 K109*/+ ). Results In overexpression models, downstream ATGs serve as alternative starting codons, generating a > 37kDa and a > 27 kDa PSEN1 C-terminal fragment (PSEN1-CTF A and PSEN1-CTF B , respectively) that retain the two catalytic aspartates of γ-secretase. Heterozygous Psen1 K109*/+ mice exhibited subtle phenotypic defects, including reduced Pen2 expression and mild APP-CTF accumulation. Notably, aged mice demonstrated significantly increased Psen2 protein expression, potentially contributing to an elevated Aβ42/Aβ38 ratio. Conclusions These findings indicate that PSEN1 c.325A > T (p.K109*) is not a complete loss-of-function mutation. However, to what extent and by what mechanism it contributes to EOAD pathogenesis remains unclear.

Bowman-Birk inhibitors (BBI) are an ancient class of serine protease inhibitors originating prior to the emergence of the angiosperms. While BBIs have been preserved in the legume (Fabaceae) and cereal (Poaceae) families, they have been lost in many other divergent lineages. However, their underlying molecular evolution and regulation of BBI remain largely uncharacterized. Our study shows that BBIs in legumes and cereals are encoded by two large and divergent gene families. BBI genes in legumes have further diversified into two subfamilies with distinct gene expression patterns. Genes in one legume BBI subfamily are specifically expressed in seeds while BBI genes in the other legume subfamily and cereal do not have significant expressions in any examined tissues including seed, root, leaf and flower. The soybean BBI gene family shows evidence of expansion via whole genome, segmental and tandem duplication. Protein sequence and structural analysis predicts that functional domains for double-headed inhibitory loops and binding abilities to trypsin and chymotrypsin are largely preserved within the soybean BBI family. The seed-specific subfamily genes are specifically expressed at maturation stages and not at embryogenesis stages. The other, non-seed BBI subfamily genes are highly responsive to a distinct spectrum of signals related to abiotic and biotic stresses. Their specific expression under non-essential biological processes for plant growth and development suggests that, although BBIs have been retained in both cereals and legumes, likely due to their role in enhancing plant fitness under natural selection pressures, they are not involved in core developmental processes. This may explain why BBIs were lost in many divergent plant lineages and support their well-established roles in plant adaptation to environmental stress. Having knocked out the seed-specific BBIs through a CRISPR/Cas9 approach, we have successfully generated soybeans which exhibited 69.4 - 73.7% reduction of trypsin inhibitor activity and 76.4 - 79.4% reduced chymotrypsin inhibitor activity. The edited soybean did not show significant changes in key agronomic traits, supporting that the functions of BBIs are not essential. While BBIs in soybean seeds may have a desirable function in natural selection, they are antinutrients from an applied perspective for their use in feed and food. It provides an opportunity to reduce BBIs in seeds for quality improvement. Our findings provide insights into molecular evolution, regulation, and function of BBI in plants, and successfully demonstrate engineering BBI in seeds to result in production of food and feed of higher nutritional value with minimal impacts on the agronomic performance of the plant.

There is a continued need for identification of novel disease drivers of acute myeloid leukemia as many patients experience relapse and have poor clinical outcomes. Analyses from our study and publicly available datasets predicted CEBPD as a novel tumor suppressor gene in acute myeloid leukemia. Consistent with the analyses, CEBPD knockdown experiments showed activation of MAPK signaling with concomitant increase in cell growth rate, while upregulation experiments suggested induction of myeloid differentiation marker CD14 expression in AML cell lines OCI-AML2 and OCI-AML5. Consistent with a previous report, our genomics analyses and azacytidine treatment experiments suggested a role for DNA methylation in downregulation of CEBPD expression during AML pathogenesis. Altogether, our results provide experimental evidence for a tumor suppressor function of CEBPD in AML.

3-Hydroxypropionic acid (3-HP) is a platform compound that can produce many chemical commodities. This study focuses on establishing and optimizing the production of 3-HP in E. coli . We constructed a series of engineered E. coli strains which can produce 3-HP via the malonyl-CoA pathway. To increase the metabolic flux of acetyl-CoA, a precursor for the synthesis of 3-HP, CRISPR/Cas9-based DNA editing technique was used to knock out the genes encoding pyruvate oxidase ( poxB) , lactate dehydrogenase ( ldhA ) and phosphate transacetylase ( pta ), thereby reducing the formation of by-products. Concurrently, the acetyl coenzyme a carboxylase gene ( accDABC ) is overexpressed on the chromosome with the objective of augmenting intracellular acetyl-CoA levels and, consequently, 3-HP production. Next, we introduced a plasmid containing a codon-optimized malonyl-CoA reductase gene ( mcr ) into the engineered strain. Finally, we constructed a transcription factor-based metabolite biosensor utilizing the PpHpdR/P hpdH system, followed by the screening of mutant strains for enhanced 3-HP production through adaptive laboratory evolution. Combining the above metabolic engineering efforts with optimisation of media and fermentation conditions, the 3-HP titer of the engineered strain WY7 increased from an initial titer 0.34 g/L to 48.8 g/L. This study encourages further research in metabolic pathway optimizationto produce 3-HP. Highlights Synthesis 3-HP in the malonyl-CoA pathway. Edit the Escherichia coli genome using the CRISPR/Cas9 system. Elevated production of 3-HP by knocking out bypass genes ldhA / pta / poxB . A biosensor was designed to respond to 3-HP concentration. Adaptive laboratory evolutionary strategies increase 3-HP production. Abstract Figure

ABSTRACT The continued development of high-dimensional CRISPR screen readouts, such as single-cell RNA sequencing and high-content imaging, necessitates compact libraries to enable functional interrogation at genome scale. Improved genome annotations yield library deprecation over time, further motivating an updated genome-wide design effort. Recently, we have developed an enhanced model, Rule Set 3, which leveraged an expansive training set and feature space to predict guide efficacy. However, the benefit of such advances to library design is limited by current approaches to balance predictions of on-target activity with off-target considerations. Here we present a guide selection strategy that identifies guides with sufficient off-target activity to justify omission from the library, thus avoiding the unnecessary exclusion of active guides. We pair this model with strategic design choices to create Jacquere, an updated, optimized, and validated Cas9 CRISPR knockout (CRISPRko) genome-wide library for the human genome.

The CRISPR-Cas system serves as an adaptive immune defence in bacteria, protecting against foreign genetic elements. In Acinetobacter baumannii , efflux pumps are major contributors to multidrug resistance (MDR). This study investigates the potential regulatory role of the CRISPR-Cas system on efflux pump genes, specifically adeB , and its association with antibiotic resistance. Methods A total of 100 clinical specimens were collected from patients admitted to the Wound Unit at Al-Hilla Teaching Hospital between March and May 2025. Standard bacteriological methods were used for isolation and identification. Antimicrobial susceptibility testing (AST) was conducted using the disk diffusion technique and interpreted according to the Clinical and Laboratory Standards Institute (CLSI) 2025 guidelines. PCR assays were used to detect the presence of CRISPR-Cas system components and the blaOXA-51 gene. Quantitative real-time PCR (qRT-PCR) was employed to assess the expression levels of the adeB efflux pump gene. Results Out of the 100 clinical samples (44 females and 55 males, aged 10–55 years), 15 (15%) isolates were confirmed as A. baumannii . AST results indicated high resistance rates to oxacillin (100%), benzylpenicillin (93.3%), erythromycin (73.3%), and tetracycline (66.7%). The isolates exhibited the highest sensitivity to tigecycline (93.3%), trimethoprim/sulfamethoxazole (93.3%), and rifampicin (86.7%). All isolates were positive for the blaOXA-51 gene.Molecular analysis revealed that the I-Fb subtype of the cas1 gene was present in 86.7% of the isolates. Expression profiling showed that adeB was overexpressed in 66.6% of the isolates. Notably, isolates harboring complete CRISPR-Cas components exhibited downregulation of adeB , suggesting a possible repressive regulatory effect of CRISPR-Cas on efflux pump expression. Conclusion This study demonstrates variability in the distribution of CRISPR-Cas elements among clinical A. baumannii isolates and suggests a potential inverse correlation between CRISPR-Cas system presence—particularly the I-Fb-cas1 subtype—and adeB efflux pump gene expression. These findings highlight the potential of CRISPR-Cas systems to modulate resistance mechanisms in A. baumannii , warranting further investigation into their therapeutic implications.

Intrinsic innate immune barriers have evolved to suppress viral infection and can reduce effective gene delivery in gene therapy. We have developed BG147, a novel cyclosporine A analogue, optimised via structure-guided design to prevent inhibition of HIV cofactor Cyclophilin A and to specifically inhibit interferon-induced transmembrane proteins (IFITM1-3). BG147 enhances VSV-G pseudotyped lentiviral vector transduction ex vivo in hematopoietic stem and progenitor cells (HSPCs) and in in vivo ocular gene therapy of photoreceptor cells in mice. Upon BG147 treatment, IFITM proteins are mislocalised and degraded through lysosomal acidification-dependent pathways. IFITM3 levels functionally return in cells 96 h after BG147 washout. BG147 promises to transform ex vivo and in vivo eye gene therapies by transiently inhibiting intrinsic immune barriers mediated by IFITM proteins to enhance a wide range of protocols. One Sentence Summary Modified cyclosporine, BG147, enhances lentivector gene therapy transduction, ex vivo in HSPC and in vivo in mouse photoreceptors, by degrading IFITM3.

Group B Streptococcus (GBS), a common colonizer of the human genital and gastrointestinal tracts, is a leading cause of neonatal bacterial meningitis, which can lead to severe neurological complications. The hypervirulent serotype III, sequence type 17 (ST-17) strain COH1 is strongly associated with late-onset disease due to its unique set of virulence factors. However, genetic manipulation of ST-17 strains is notoriously challenging, limiting the ability to study key pathogenic genes. In this study, we developed a CRISPR interference (CRISPRi) system utilizing an endogenous catalytically inactivated Cas9 (dCas9) in the COH1 strain, enabling targeted and tunable gene expression knockdown. We confirmed the efficacy of this system through hemolysis assays, qPCR transcriptional analysis, and in vitro infection models using human brain endothelial cells. The CRISPRi system successfully produced phenotypic knockdowns of essential virulence genes, including pilA, srr2 , and iagA , reducing adhesion, invasion, and inflammatory responses at the blood-brain barrier. This platform enables rapid gene knockdowns for functional genomics in ST-17 GBS, enabling high-throughput screening and pathogenesis research. Importance Group B Streptococcus (GBS) remains the world’s leading cause of neonatal meningitis. GBS-host interactions at the blood-brain barrier (BBB) are dependent on bacterial factors, including surface factors and two-component systems. Multi-locus sequence type 17 (ST-17) GBS strains are highly associated with neonatal meningitis, and these strains harbor many virulence factors for infection at the BBB. Historically, these factors have been studied using traditional knockout mutagenesis, which has proven challenging in the most common ST-17 lab strain, COH1. This study utilizes CRISPR interference (CRISPRi) to generate rapid expression knockdown. This study validates a CRISPRi-enabled COH1 dCas9 strain as a versatile tool for probing GBS pathogenesis at the BBB.

Programmable DNA integration using CRISPR-associated transposons (CASTs) offers powerful capabilities for genome engineering. The single effector Cas12k CAST examples evolved from a fixed guide TnpB nuclease protein. Here, we engineer de novo RNA-guided transposition systems, where the single guide RNA effector components are repurposed nuclease-dead TnpB-family proteins. These compact systems mediate high-efficiency guide RNA-directed DNA insertion with preserved orientation control and target immunity, reduced off-site targeting, release of a host factor requirement, and can be paired with an exonuclease domain to mediate cut-and-paste transposition. In this engineered context, the TnpB derivatives show features not predicted from the original enzymes suggesting untapped avenues for improvement. In parallel, we show that mutations at the TniQ-TnsC interface in the Cas12k CAST system selectively attenuate off-site insertions while enhancing on-site activity. These results establish how Cas12 proteins and antecedent TnpB proteins can be engineered for high performance and specificity with guide RNA directed systems.

Loeys-Dietz Syndrome type 3 (LDS3) is caused by pathogenic (P)/likely pathogenic (LP) variants in the SMAD3 gene and is characterized by aneurysm formation and arterial tortuosity, which can lead to life-threatening complications. There is an unmet need for suitable cell models to study LDS3 at a cellular and molecular level. Induced pluripotent stem (iPS) cells offer a promising approach because they can be genetically modified using CRISPR/Cas9 technology and differentiated into disease-relevant cell types. As it is difficult to obtain aortic vascular smooth muscle cells (VSMCs) from patients, iPS cells differentiated into VSMCs provide an ideal model to study cellular aneurysmal phenotypes. In this study, we generated iPS cell models carrying (P/LP) SMAD3 variants. These cell models were generated either by using CRISPR/Cas9 mediated introduction of indels and deletions to introduce SMAD3 variants, or by reprogramming of fibroblasts derived from SMAD3 patients. These iPS cell lines were characterized for SMAD3 expression by Western blotting and validated for pluripotency through immunofluorescence and qPCR. Moreover, the patient-derived iPS cell lines were shown to differentiate into smooth muscle cells (SMCs), which are relevant to study the molecular mechanisms underlying aneurysm formation in LDS3 patients. Our findings highlight the potential of these iPS-based models to investigate the pathophysiology of LDS3 and facilitate the development of therapeutic strategies for aortic aneurysms.