Antimicrobial resistance (AMR) threatens global health. In this manuscript, I review recent literature underscoring the promise of engineered bacteriophages and CRISPR-Cas systems as targeted strategies against resistant bacteria. These approaches offer alternatives to broad-spectrum antibiotics by precisely disrupting biofilms and inactivating resistance genes—whether applied independently or in tandem. I also underscore the essential role of public-private partnerships in advancing clinical applications and catalyzing the translation of innovative research into practice

CD19-CAR-T-cells emerge as a major therapeutic option for relapsed/refractory B-cell-derived malignancies, however approximately half of patients eventually relapse. To identify resistance-driving factors, we repeatedly exposed B-cell lymphoma/B-cell acute lymphoblastic leukemia to 4-1BB/CD28-based CD19-CAR-T-cells in vitro . Generated models revealed costimulatory domain-dependent differences in CD19 loss. While CD19-4-1BB-CAR-T-cells induced combination epitope/total CD19 protein loss, CD19-CD28-CAR-T-cells did not drive antigen-escape. Consistent with observations in patients relapsing after CD19-4-1BB-CAR-T-cells, we identified CD19 frameshift/missense mutations affecting residues critical for FMC63 epitope recognition. Mathematical simulations revealed that differences between CD19-4-1BB- and CD19-CD28-CAR-T-cells activity against low-antigen-expressing tumor contribute to heterogeneous therapeutic responses. By integrating in vitro and in silico data, we propose a biological scenario where CD19-4-1BB-CAR-T-cells fail to eliminate low-antigen tumor cells, fostering CAR-resistance. These findings offer mechanistic insight into the observed clinical differences between axi-cel (CD28-based) and tisa-cel (4-1BB-based)-treated B-cell lymphoma patients and advance our understanding on CAR-T resistance. Furthermore, we underscore the need for specific FMC63 epitope detection to deliver information on antigen levels accessible for CD19-CAR-T-cells. Visual abstract

ABSTRACT Viruses have evolved elaborate mechanisms to hijack the host mRNA translation machinery to direct viral protein synthesis. Picornaviruses, whose RNA genomes lack a cap structure, inhibit cap-dependent mRNA translation, and utilize an internal ribosome entry site (IRES) in the RNA 5′-UTR to recruit the 40S ribosomal subunit. IRES activity is stimulated by a set of host proteins termed IRES trans -acting factors (ITAFs). The cellular protein ITAF 45 (also known as PA2G4 and EBP1) was identified as an essential ITAF for foot-and-mouth disease virus (FMDV), with no apparent role in cell-free systems for the closely related viruses harboring similar IRES elements such as encephalomyocarditis virus (EMCV) and Theiler’s murine encephalomyelitis virus (TMEV). Here, we demonstrate that ITAF 45 is a pervasive host factor within cells for picornaviruses containing a Type II IRES. CRISPR/Cas9 knockout of ITAF 45 in several human cell lines conferred resistance to infection with FMDV, EMCV, TMEV, and equine rhinitis A virus (ERAV). We show that ITAF 45 enhances initiation of translation on type II IRESs in cell line models. This is mediated by the C-terminal lysine-rich region of ITAF 45 known to enable binding to viral RNA. These findings challenge previous reports of a unique role for ITAF 45 in FMDV infection, positioning ITAF 45 as a promising antiviral target for various animal viruses and emerging human cardioviruses.

We present an open source, 3D-printed toolbox for avian embryology. The toolbox includes an electroporation chamber for transfecting functional molecular reagents into developing embryos, and a set of live-imaging chambers, which support avian embryo development while presenting them to a wide variety of microscope setups. We demonstrate both electroporation and imaging chambers by transfecting novel fluorescent reporter constructs for the TGF-beta signalling pathway and Pax7, Brachyury, Cdx2 and Sox2:Oct4 transcription factors into chick embryos and performing time-lapse imaging from stages HH3 - HH12 via both widefield and confocal fluorescence microscopy. Open-source code and ready-to-print STL files are freely available from a GitHub repository in line with FAIR (findability, accessibility, interoperability, and reusability) principles.

Cytoplasmic FMRP Interacting Protein 2 (CYFIP2) a component of the Wave Regulatory Complex (WRC), one of the most important players in regulating cellular actin dynamics. Interestingly, CYFIP2 transcript undergoes RNA editing, an epitranscriptomic modification catalysed by ADAR enzymes, that leads adenosine (A) to inosine (I) deamination. CYFIP2 editing in the coding sequence results in a K/E substitution at amino acid 320. The functional meaning of this regulation is still unknown. In this study, we aim at investigating the potential implication of CYFIP2 RNA editing related to actin dynamics during cell differentiation, axon development and synaptogenesis in neural cells. We have generated SH-SY5Y neuroblastoma cell lines in which CYFIP2 gene has been functionally inactivated by CRISPR-Cas9 technology. CYFIP2 KO cells showed profound actin filaments disorganisation and loss of the capability to differentiate into a neuronal-like phenotype. Overexpression of both CYFIP2 unedited (K) and edited (E) isoforms rescued normal capability. Finally, we took advantage of primary neuronal culture where endogenous CYFIP2 was knocked down by shRNA technology and CYFIP2 editing variants were overexpressed. While CYFIP2 KD cells reported a decrease in axon development and spine frequency, CYFIP2-E variants increase the number of axon branches, total axon length and dendritic spine frequency compared to either CYFIP2 KD cells or CYFIP-K variants. Overall, our work reveals for the first time a functional significance of the CYFIP2 K/E RNA editing process in regulating the spreading of neuronal axons during the initial stages of in-vitro development and the process of spinogenesis.

Abstract The CRISPR-associated endonuclease Streptococcus pyogenes Cas9 (SpCas9) enables site-specific DNA cleavage by transitioning from a pre-catalytic conformation to a catalytically active state, yet how its HNH catalytic domain undergoes an approximately 40 Å displacement towards the target DNA has remained elusive. Here, we combined extensive unbiased molecular dynamics simulations, spanning a cumulative timescale of 160 µs, with Markov state modeling to map the kinetic pathway of SpCas9 activation. In vitro DNA cleavage assays and a cellular fluorescence reporter system further validated the atomic-level mechanisms revealed by our simulations. We found that the folding of the L1 linker and unfolding of the L2 linker serve as the principal driving force, inducing a “gear-and-wedge” cooperative motion within the HNH domain. Concurrently, the REC2 domain moved outward to accommodate the displaced HNH domain and formed transient stabilizing interactions with the HNH domain along the activation route. Site-directed mutagenesis of key L2 linker residues and REC2 loops markedly reduced SpCas9 cleavage efficiency in both HEK293T cells and biochemical assays, underscoring their critical role in SpCas9 ribonucleoprotein activation. Collectively, this study provides a high-resolution view of SpCas9 catalytic activation and opens up new avenues for the rational design of SpCas9 variants with enhanced performance and specificity.

The severe acquired respiratory coronavirus–2 (SARS–CoV-2) infection has initiated both acute and chronic COVID–19 disease between 2020 and 2023, currently evolving with other homologous prior coronavirus strains of the Nidoviridae order, which encompasses other prevalent alpha/ beta coronaviruses, but also the Middle East Respiratory Syndrome (MERS-CoV) and SARS-CoV-1, with recent SARS–CoV–2 variants, increasing demands for effective immunogens and therapeutic approaches that will reduce global disease burden and further infection from SARS–CoV-2 affected individuals that may experience post acute sequelae (PASC) or “Long COVID”. Following a worldwide programme of prophylactic vaccination, there is still a dilemma in the efforts to find prophylactic and early therapeutic approaches that would treat novel SARS-CoV-2 variants and prevent future epidemics or pandemics within host human and animal populations, where zoonotic or cross species transfer naturally occurs. Concerns about viral immune escape intersect at a specific point; a gained evolutionary ability of several viruses to co–infect and compete against previous scientific advances since 1796 that remain undetected or asymptomatic during the early stages of infection progressing to symptomatic and severe disease via the double methylation of the 5' end of eukaryotic DNA or RNA-based viral genomes, the 7-MeGpppA2’-O-Me cap, and its double methylation capping process is performed by the activated viral 2’ - O - Methyltransferase (MTase) enzyme, a complex of two viral non-structural proteins (NSPs) joined together through an activation process (NSP10/16) and by N7-Methyltransferase (N7-MTase/NSP14), respectively. Moreover, it was discovered that polymorphic viruses translate NSP1, which prevents the activation of various Pattern Recognition Receptors (PRRs), and consequently, detection of Pathogen-Associated Molecular Patterns (PAMPs) and Damage-Associated Molecular Patterns (DAMPs) alike. NSP1 also silences important interferon-encoding genes (INGs) and interferon-stimulated genes (ISGs), is signalled in a paracrine manner to neighbouring cells, and that induces the apoptosis of host cells, inducing an effect of “trace erase” effect and making the viral infection as immunologically “invisible” as possible during the initial, key stages of viral replication and distribution, all such mechanisms occurring independently of the viruses in cause. Another important viral NSP is NSP14, as it plays two functional roles that are independent of each other; to produce new viral genetic material for the purpose of maintaining the validity of the viral genome as well, and not just transfer a methyl group to the 5’ end of the viral genome. Other viral NSPs share a role with NSP1, 10, 14 and 16 in directly suppressing the activation of PRRs and ISGs, and all such viral proteins help the virus in its process of self-camouflaging against first- and second-line immunity, thereby often severely impacting the quality of the produced adaptive immune responses. The outcome of all such phenomena is the sharp decrease in the host Type I and Type III interferons' (IFNs) rate of synthesis by the host cells, that would usually occur and affect homeostatic cellular pathways, resulting in further viral replication and induced apoptosis. Nonetheless, effects of microbial immune evasion during the development of other viral or carcinogenic pathologies are not widely known. In short, polymorphic viruses developed a proportionate evolutionary response against developed adaptive immune responses, by currently relying on gaps mostly situated in the natural immune system in their process of molecular self-camouflaging. Scientists developed numerous approaches of early treatment that generally showed good success rates and fewer risks of adverse events, and the still early present stages of COVID-19 research should also be taken into consideration whilst filtering for the most appropriate solutions. For example, the administration of recombinant human interferons I and III into the nasal mucosa cellular layer, as key mediators of anti–viral activity, can simulate intracellular infection and stimulate cellular activity in a timely manner, training the innate and adaptive immune system cells to develop and appropriately stimulate an adequate immune response through B and T cells. Another example could involve the treatment of natural and adaptive lymphocytes with a low dose of IFNs I and possibly III, prior to their insertion into the host lymphatic system, possibly alongside additional recruitment of plasmacytoid dendritic cells (pDCs) as further interferon “factories”, all with the purpose of early infection management. It might be that focusing on directly offering the immune system the information about the genetics and protein structure of the pathogen, rather than training its first-line mechanisms to develop faster, excessively increases its specificity, making it reach a level that brings the virus the opportunity to evolve and escape previously-developed host immune mechanisms. It is until the scientific community realises this potentially crucial aspect that large proportions of the world population will probably continue to face serious epidemics and pandemics of respiratory diseases over the coming several decades, evidenced with dengue fever and more recently, monkeypox and possibly avian flu. Of note, it has been indicated that IFN I and / or III display significant immunising, early therapeutic and clinical disease onset-attenuating effects for many other microbial evoked diseases, as well as for a number of oncological diseases. Microbial agents could undergo loss-of-function research upon genes responsible for inducing clinical illness whilst keeping genes responsible for microbial reproduction and transmission at least generally as functional, CRISPR-Cas9 genome editing to have genes encoding proteins suppressive of the host interferon system eliminated prior to human genes encoding Pattern Recognition Receptor activator or agonist proteins, such as outer membrane proteins of Neisseria meningitidis, as well as Type I, Type III and possibly even Type IV Interferons and various ISGs inserted into the microbial genome. Such an approach would be based upon the model of editing genes of harmless bacteria to transform such them into “producers” and “distributors” of human insulin, and could turn several microbial agents into clinically harmless, transmissible “factories” for various key elements of the host interferon system, potentially placing such microbes into a reverse evolutionary path that would be deemed as “natural de-selection”, visibly reducing the average burden of disease and metabolic stresses, which in turn could gradually increase average human and animal lifespans worldwide.

ABSTRACT Gene drives are selfish genetic elements which promise to be powerful tools in the fight against vector-borne diseases such as malaria. We previously proposed population replacement gene drives designed to better withstand the evolution of resistance by homing through haplolethal loci. Because most mutations in the wild-type allele that would otherwise confer resistance are lethal, only successful drive homing permits the cell to survive. Here we outline the development and characterization of two ΦC31-Recombination mediated cassette exchange (RMCE) gene drive docking lines with these features in Anopheles gambiae , a first step towards construction of robust gene drives in this important malaria vector. We outline adaption of the technique HACK (Homology Assisted CRISPR knockin) to knock-in two docking site sequences into a paired haplolethal-haplosufficient (Ribosome-Proteasome) locus, and confirm that these docking lines permit insertion of drive-relevant transgenes. We report the first anopheline proteasome knockouts, and identify ribosome mutants that reveal a major hurdle that such designs must overcome to develop robust drives in the future. Although we do not achieve drive, this work provides a new tool for constructing future evolution-robust drive systems and reveals critical challenges that must be overcome for future development of gene drives designed to target haplolethal loci in anophelines and, potentially, other metazoans.

Summary Sex chromosomes shape male (XY) - female (XX) differences in development and disease. These differences can be modelled in vitro by comparing XY and XX human induced pluripotent stem cells (hiPSCs). However, in this system, inter-individual autosomal variation and unstable X-dosage compensation can confound identification of sex chromosomal effects. Here, we utilise sex chromosome loss in XXY fibroblasts to generate XX and XY hiPSCs that are autosomally isogenic and exhibit stable X-dosage compensation. We also create X-monosomic (XO) hiPSCs, to investigate X-Y dosage effects. Using these autosomally isogenic lines, we examine sex differences in pluripotent stem cell expression. Transcriptional differences between XX and XY hiPSCs are surprisingly modest. However, X-haploinsufficiency induces transcriptional deregulation predominantly affecting autosomes. This effect is mediated by Y-genes with broad housekeeping functions that have X-homologues escaping X-inactivation. Our isogenic hiPSC lines provide a resource for exploring sex chromosome effects on development and disease in vitro .

ABSTRACT Tuberculosis (TB) and COVID-19 are leading infectious diseases with high mortality, caused by Mycobacterium tuberculosis ( Mtb ) and SARS-CoV-2 (SC2) , respectively. Co-infection is common but is often undiagnosed as it is challenging to process both pathogens from a single sample. In this study, we present a simple and efficient method for co-extracting nucleic acids (NA) from these two distinct respiratory pathogens for downstream diagnostic testing. We evaluated three different nucleic acid amplification (NAA)-based platforms, LightCycler480 (LC480) qPCR, Qiacuity digital PCR (dPCR), and Cytation3 for CRISPR-Cas13a-based SHINE-TB/SC2 detection assays. Chelex-100 chelating resin-based boiling preparation method was optimized for Mtb NA extraction from saliva and sputum. Saliva showed compatibility with all three platforms, with sensitivity as low as 100 CFU/ml (or 2 genomic copies/µl). This method worked well for sputum using dPCR at 100% (21/21) positivity, though the CRISPR-based SHINE-TB assay showed more variability and sensitivity to sputum inhibitor carry-over, resulting in an 81% positive rate (17/21). Diluting sputum with TE buffer (1:1) improved the detection (2/4). Extraction efficiency of our method was 48%, 62.2%, 86.4% and 99.3% for concentrations 10 5 , 10 4 , 10 3 and 10 CFU/ml, respectively. The dynamic range for Mtb spiked in pooled sputum showed 100% detection (N=8) at ≥10 3 CFU/ml with all three methods. Dual-pathogen co-extraction and detection of SC2 (10 5 PFU/ml) and Mtb (10 5 CFU/ml) in salivary sputum was successful using CRISPR-Cas13a assays. We have developed a rapid and efficient co-extraction method for multi-pathogen testing across diagnostic platforms and believe this is the first protocol optimized to co-extract Mtb and SARS-CoV-2 from a single sample.

The emergence of multidrug-resistant and extensively drug-resistant Mycobacterium tuberculosis strains poses serious challenges to global tuberculosis control, highlighting the urgent need to unravel the mechanisms underlying multidrug resistance. In this study, we screened for spontaneous bortezomib (BTZ)-resistant mutants of Mycobacterium smegmatis and identified a strain, Msm-R1-2, which exhibited high-level resistance to both BTZ and linezolid. Whole-genome sequencing revealed mutations in MSMEG_1380 and MSMEG_0965 genes, encoding a transcriptional regulator (involved in regulating efflux pump expression) and a porin, respectively are potential contributors to drug resistance. CRISPR-Cpf1-assisted gene knockout and editing experiments confirmed that dual mutations in MSMEG_1380 and MSMEG_0965 synergistically enhanced resistance to BTZ and LZD, conferring cross-resistance to other antibiotics, including moxifloxacin and clofazimine. Ethidium bromide accumulation assay demonstrated that mutations in MSMEG_0965 reduce cell wall permeability, contributing to multidrug resistance. Furthermore, previous studies have shown that mutations in MSMEG_1380 upregulate the mmpS5-mmpL5 efflux system, thereby promoting drug efflux and reducing intracellular drug concentrations, while mutations in MSMEG_0965 impair porin function, limiting antibiotic uptake and significantly contributing to the multidrug-resistant phenotype. Collectively, these findings provide valuable insights into the molecular mechanisms of mycobacterial drug resistance, underscoring the pivotal roles of efflux and uptake pathways in the development of multidrug resistance.

Understanding how cellular pathways interact is crucial for treating complex diseases like cancer, yet our ability to map these connections systematically remains limited. Individual gene-gene interaction studies have provided insights 1,2 , but they miss the emergent properties of pathways working together. To address this challenge, we developed a multi-gene approach to pathway mapping and applied it to CRISPR data from the Cancer Dependency Map 3 . Our analysis of the electron transport chain revealed certain blood cancers, including acute myeloid leukemia (AML), depend on an unexpected link between Complex II and purine metabolism. Through stable isotope metabolomic tracing, we found that Complex II directly supports de novo purine biosynthesis and exogenous purines rescue AML from Complex II inhibition. The mechanism involves a metabolic circuit where glutamine provides nitrogen to build the purine ring, producing glutamate that Complex II must oxidize to sustain purine synthesis. This connection translated to a metabolic vulnerability whereby increasing intracellular glutamate levels suppresses purine production and sensitizes AML to Complex II inhibition. In mouse models, targeting Complex II triggered rapid disease regression and extended survival in aggressive AML. The clinical relevance of this pathway emerged in human studies, where higher Complex II gene expression correlates with both resistance to mitochondria-targeted therapies and worse survival outcomes specifically in AML patients. These findings establish Complex II as a central regulator of de novo purine biosynthesis and identify it as a promising therapeutic target in AML.

CRISPR/Cas9 is a powerful tool for targeted genome engineering experiments. With CRISPR/Cas9, genes can be deleted or modified by inserting small peptides, fluorescent proteins or other tags for protein labelling experiments. Such experiments are important for detailed protein characterization in vivo . However, designing and cloning the corresponding constructs can be repetitive, time consuming and laborious. To aid users in CRISPR/Cas9-based genome engineering experiments, we built CrisprBuildr, a web-based application that allows users to delete genes or insert fluorescent proteins at the N- or C-terminus of their gene of choice. The application is built on the Drosophila melanogaster genome but can be used as a template for other available genomes. We have also generated new tagging vectors, using EGFP and mCherry combined with the small peptide SspB-Q73R for use in iLID-based optogenetic experiments. CrisprBuildr guides users through the process of designing guide RNAs and repair template vectors. CrisprBuildr is an open-source application and future releases could incorporate additional tagging or deletion vectors, genomes or CRISPR applications.

Plasma cell subsets vary in their lifespans and ability to sustain humoral immunity. We conducted a genome-wide CRISPR-Cas9 screen in a myeloma cell line for factors that promote surface expression of CD98, a marker of longevity in primary mouse plasma cells. A large fraction of genes found to promote CD98 expression in this screen are involved in secretory and other vesicles, including many subunits of the V- type ATPase complex. Chemical inhibition or genetic ablation of V-type ATPases in myeloma cells reduced antibody secretion. Primary mouse and human long-lived plasma cells had greater numbers of acidified vesicles than did their short-lived counterparts, and this correlated with increased secretory capacity of IgM, IgG, and IgA. The screen also identified PI4KB, which promoted acidified vesicle numbers and secretory capacity, and DDX3X, an ATP-dependent RNA helicase, the deletion of which reduced immunoglobulin secretion independently of vesicular acidification. Finally, we report a plasma-cell intrinsic function of the signaling adapter MYD88 in both antibody secretion and plasma cell survival in vivo . These data reveal novel regulators of plasma cell secretory capacity, including those that also promote lifespan. One Sentence Summary Long-lived plasma cells rely on V-type ATPases, PI4K, DDX3X, and MYD88 signals for maximal secretory capacity

Background Colon cancer progression heavily relies on intricate mechanisms of invasion, metastasis, and migration. Tight junction protein Cldn2 has emerged as a potential regulator of these processes. This study aimed to elucidate the molecular mechanisms linking Clan2 deletion to gene expression changes related to motility, invasion, and metastasis in colon caner. Methods CRISPR/Cas9-mediated knockout of human Cldn2 in HCT116 cells was conducted, and the resulting cells were compared to the wild-type cells using real-time PCR to analyze the expression of genes associated with invasion and metastasis. Results Cldn2-KO resulted in a widespread downregulation of genes linked to motility, invasion, and metastasis, including ZONAB, NDRG1, Cldn14, Cldn23, Bcl2, , P53, and BCL-6. These findings suggest a potential regulatory role of Cldn2 in the expression of these genes, influencing colon cancer cell migration and spread. Conclusion This study identified Claudin-2 as a crucial regulator of genes involved in colorectal cancer metastasis. Downregulation of these genes upon Claudin-2 deletion suggests its inhibitory role in cancer cell motility and invasion. Further investigation into the specific downstream signaling pathways mediated by Claudin-2 could pave the way for novel therapeutic strategies targeting metastasis inhibition.

ABSTRACT Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-associated proteins (Cas) systems have revolutionized genome editing by providing high precision and versatility. However, most genome editing applications rely on a limited number of well-characterized Cas9 and Cas12 variants, constraining the potential for broader genome engineering applications. In this study, we extensively explored Cas9 and Cas12 proteins and developed CasGen, a novel transformer-based deep generative model with margin-based latent space regularization to enhance the quality of newly generative Cas9 and Cas12 proteins. Specifically, CasGen employs a strategies that combine classification to filter out non-Cas sequences, Bayesian optimization of the latent space to guide functionally relevant designs, and thorough structural validation using AlphaFold-based analyses to ensure robust protein generation. We collected a comprehensive dataset with 3,021 Cas9, 597 Cas12, and 597 Non-Cas protein sequences from reputable biological databases such as InterPro and PDB. To validate the generated proteins, we performed sequence alignment using the BLAST tool to ensure novelty and filter out highly similar sequences to existing Cas proteins. Structural prediction using AlphaFold2 and AlphaFold3 confirmed that the generated proteins exhibit high structural similarity to known Cas9 and Cas12 variants, with TM-scores between 0.70 and 0.85 and root-mean-square deviation (RMSD) values below 2.00 Å. Sequence identity analysis further demonstrated that the generated Cas9 orthologs exhibited 28% to 55% identity with known variants, while Cas12a variants show up to 48% identity. Our results demonstrate that the proposed Cas generative model has significant potential to expand the genome editing toolkit by designing diverse Cas proteins that retain functional integrity. The developed deep generative approach offers a promising avenue for synthetic biology and therapeutic applications, enableling the development of more precise and versatile Cas-based genome editing tools.

Clustered regularly interspaced palindromic repeat (CRISPR)/CRISPR-associated protein (Cas)-based in vivo chromosomal rearrangements are a promising approach for generating model organisms with specific chromosomal abnormalities. However, conventional in vivo methods rely on viral vectors, which are expensive, require specialized equipment, and pose potential safety risks, thereby limiting their widespread application. To overcome the limitations above, we developed a novel, efficient, and cost-effective in vivo chromosomal engineering strategy using CRISPR ribonucleoprotein electroporation for the murine uterine epithelium. Our method successfully induced translocations at multiple loci and repaired a 57.8-Mb inversion. The findings of the present study establish in vivo electroporation as a practical alternative to traditional chromosomal engineering methods and provide a foundation for its broader application in genome editing technologies.

Abstract Klebsiella pneumoniae (Kp) has evolved as a major public health threat due to its multidrug-resistance (MDR) and hypervirulence. Current genome-editing tools for Kp are constrained by cumbersome workflows, low flexibility, and limited scalability. Here, we present the RECKLEEN system —Recombineering/CRISPR-based KLebsiella Engineering for Efficient Nucleotide editing — as a single plasmid platform designed for precise genetic manipulation of Kp. RECKLEEN combines lambda Red recombineering with powerful CRISPR-Cas9-based targeted counterselection, achieving up to 99.998% killing efficiency. By implementing the near PAM-less SpG Cas9 variant in RECKLEEN, the compatible target sequence spectrum was significantly broadened. This approach enables deletions, point mutations, and DNA integrations, with efficiencies reaching 100% of the counter-selected clones. Simultaneous multi-target deletions were accomplished with up to 72% efficiency. To streamline the process, we developed a toolbox of eleven plasmids based on a modular cloning standard, enabling time- and resource-efficient assembly of editing constructs. This allows a 5-days workflow, from plasmid construction to the generation of strains with the desired genetic modification(s). The efficacy of RECKLEEN extends to various MDR Kp strains, such as ATCC 700721, ATCC BAA-1705, and ATCC 700603, demonstrating its broad applicability. RECKLEEN significantly enhances genome-editing capabilities for Kp, advancing research into its pathology and MDR mechanisms.

Polygenic traits are expected to show high genetic redundancy and therefore low repeatability in the genomic response to selection. We tested this prediction by selecting for large body size in the black soldier fly ( Hermetia illucens ). Over three replicate experiments selected for large body size, we found a strong and repeatable phenotypic response, with a mean 15% increase in body size. Selected lines also increased in larval growth rate (+19%) and average protein content (+14%), suggesting that selection on large body size does not result in strong trade-offs. In contrast to the predictability of the phenotypic response across replicates, whole genome sequencing identified a highly polygenic and non-repeatable genomic response. We identified 120, 301 and 157 outlier genomic regions in the three replicates, but high redundancy with only four shared regions. Among 12 candidate genes found in these regions, the insulin-like receptor gene ( HiInR ) was confirmed as regulating larval growth using a CRISPR knockout experiment. In summary, polygenic quantitative traits show high genetic redundancy, even where the phenotypic response to selection is highly repeatable.

Escherichia coli is a ubiquitous gut commensal but also an opportunistic pathogen responsible for severe intestinal and extra-intestinal infections. Shiga toxin-producing E. coli (STEC) pose a significant public health threat, particularly in children, where infections can lead to bloody diarrhea and progress to hemolytic uremic syndrome (HUS), a life-threatening condition with long-term complications. Antibiotics are contraindicated in STEC infections due to their potential to induce prophages carrying Shiga toxin ( stx) genes, triggering toxin production. Here, we present a CRISPR-based antimicrobial strategy that selectively targets and eliminates O157 STEC clinical isolates while preventing toxin release. We designed a Cas12 nuclease to cleave >99% of all stx variants found in O157 strains, leading to bacterial killing and suppression of toxin production. To enable targeted delivery, we engineered a bacteriophage-derived capsid to specifically transfer a non-replicative DNA payload to E. coli O157, preventing its dissemination. In a mouse STEC colonization model, our therapeutic candidate, EB003, reduced bacterial burden by a factor of 3×10 3 . In an infant rabbit disease model, EB003 mitigated clinical symptoms, abrogated stx-mediated toxicity, and accelerated epithelial repair at therapeutically relevant doses. These findings demonstrate the potential of CRISPR-based antimicrobials for treating STEC infections and support further clinical development of EB003 as a precision therapeutic against antibiotic-refractory bacterial pathogens.

The rate, spectrum, and biases of mutations represent a fundamental force shaping biological evolution. Convention often attributes oxidative DNA damage as a major driver of spontaneous mutations. Yet, despite the contribution of oxygen to mutagenesis and the ecological, industrial, and biomedical importance of anaerobic organisms, relatively little is known about the mutation rates and spectra of anaerobic species. Here, we present the rates and spectra of spontaneous mutations assessed anaerobically over 1000 generations for three fermentative lactic acid bacteria species with varying levels of aerotolerance: Lactobacillus acidophilus, Lactobacillus crispatus, and Lactococcus lactis. Our findings reveal highly elevated mutation rates compared to the average rates observed in aerobically respiring bacteria with mutations strongly biased towards transitions, emphasizing the prevalence of spontaneous deamination in these anaerobic species and highlighting the inherent fragility of purines even under conditions that minimize oxidative stress. Beyond these overarching patterns, we identify several novel mutation dynamics: positional mutation bias around the origin of replication in Lb. acidophilus, a significant disparity between observed and equilibrium GC content in Lc. lactis, and repeated independent deletions of spacer sequences from within the CRISPR locus in Lb. crispatus providing mechanistic insights into the evolution of bacterial adaptive immunity. Overall, our study provides new insights into the mutational landscape of anaerobes, revealing how non-oxygenic factors shape mutation rates and influence genome evolution.

Investigating the temporal dynamics of gene expression is crucial for understanding gene regulation across various biological processes. The Fluorescent Timer protein (Timer) offers a valuable tool for such studies, exemplified by our Foxp3 Timer-of-cell- kinetics-and-activity (Tocky), which facilitates the analysis of Foxp3 dynamics at the single-cell level. However, the complexity of Timer fluorescence profiles has limited its application, necessitating novel analytic approaches. Here, we introduce an integrative method that combines molecular biology with machine learning (ML) to elucidate enhancer-specific regulation of transcriptional dynamics. Our approach is demonstrated by mutating the enhancer, Conserved Non-coding Sequence 2 (CNS2), of the Foxp3 Timer transgene in Foxp3-Tocky embryos via CRISPR. Technologically, we have developed machine learning tools that employ Random Forest and Convolutional Neural Networks with image conversion techniques, including Gradient-weighted Class Activation Mapping. These independent ML models effectively elucidated CNS2-specific regulation of Foxp3 transcription and underscored the roles of CNS2 in regulating the autoregulatory loop of Foxp3 transcription. In conclusion, our study reveals previously unrecognized roles of CNS2 in Foxp3 transcriptional dynamics, showcasing the potential of the CRISPR Tocky assay as an advanced method to understand transcriptional dynamics in vivo.

Summary A unique feature of temperate phages is the ability to protect their host bacteria from a second phage infection. Such protection is granted at the lysogenic state, where the phages persist as prophages integrated within the bacterial chromosome, expressing genes that defend the host and themselves from predation. Here, we report a prophage-encoded anti-phage defense system that inhibits DNA packaging of invading phages in Listeria monocytogenes . This system includes a defense protein, TerI, and two self-immunity proteins, anti-TerI1 and anti-TerI2. TerI targets the terminase complex of invading phages to prevent DNA translocation into procapsids without halting the lytic cycle, leading to the release of unpacked non-infectious procapsids upon bacterial lysis. In contrast, the self-immunity proteins, anti-TerI1 and anti-TerI2, counteract TerI during prophage induction to allow virion production. This unique prophage-encoded anti-phage defense system, TERi, is prevalent in Listeria phages, providing population-level host protection without compromising the prophage lytic lifecycle.

Despite the large variety of insect species with divergent morphological, developmental and physiological features questions on gene function could for a long time only be addressed in few model species. The adoption of the bacterial CRISPR-Cas system for genome editing in eukaryotic cells widened the scope of the field of functional genetics: for the first time the creation of heritable genetic changes had become possible in a very broad range of organisms. Since then, targeted genome editing using the CRISPR-Cas technology has greatly increased the possibilities for genetic manipulation in non-model insects where molecular genetic tools were little established. The technology allows for site-specific mutagenesis and germline transformation. Importantly, it can be used for the generation of gene knock-outs, and for the knock-in of transgenes and generation of gene-reporter fusions. CRISPR-Cas induced genome editing can thus be applied to address questions in basic research in various insect species and other study organisms. Notably, it can also be used in applied insect biotechnology to design new pest and vector control strategies such as gene drives and precision guided Sterile Insect Technique. However, establishing CRISPR in a new model requires several practical considerations that depend on the scientific questions and on the characteristics of the respective study organism. Therefore, this review is intended to give a literature overview on different CRISPR-Cas9 based methods that have already been established in diverse insects. After discussing some required pre-conditions of the study organism, we provide a guide through experimental considerations when planning to conduct CRISPR-Cas9 genome editing, such as the design and delivery of guide RNAs, and of Cas9 endonuclease. We discuss the use of different repair mechanisms including homology directed repair (HDR) for a defined insertion of genetic elements. Furthermore, we describe different molecular methods for genetic screening and the use of visible markers. We focus our review on experimental work in insects, but due to the ubiquitous functionality of the CRISPR-Cas system many considerations are transferable to other non-model organisms.

Background Colorectal cancer (CRC) progression from adenoma to adenocarcinoma is associated with global reduction in 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC). DNA hypomethylation continues upon liver metastasis. Here we examine 5hmC changes upon progression to liver metastasis. Results 5hmC is increased in metastatic liver tissue relative to the primary colon tumour and expression of TET2 and TET3 is negatively correlated with risk for metastasis in patients with CRC. Genes associated with increased 5-hydroxymethylcytosine show KEGG enrichment for adherens junctions, cytoskeleton and cell migration around a core cadherin (CDH2) network. Overall, the 5-hydroxymethylcyosine profile in the liver metastasis is similar to normal colon appearing to recover at many loci where it was originally present in normal colon and then spreading to adjacent sites. The underlying sequences at the recover and spread regions are enriched for SALL4, ZNF770, ZNF121 and PAX5 transcription factor binding sites. Finally, we show in a zebrafish migration assay using SW480 CRISPR-engineered TET knockout and rescue cells that reduced TET expression leads to a reduced migration frequency. Conclusion Together these results suggest a biphasic trajectory for 5-hydroxymethyation dynamics that has bearing on potential therapeutic interventions aimed at manipulating 5-hydroxymethylcytosine levels.