Nutritional immunity is an antimicrobial strategy that evolved to starve pathogens of essential nutrients, with death as the desired outcome. Here, we report that transient iron starvation of the obligate intracellular pathogen Chlamydia trachomatis , growing in endocervical epithelial cells, enhances pathogen recognition by the host cell through the dysregulation of a peptidoglycan (PG) remodeling enzyme, resulting in the activation of the nucleotide-binding oligomerization domain 2 (NOD2) pathway that recognizes PG fragments, increased production of tumor necrosis factor alpha (TNF) via increased activation of NF-κB, which correlated with death of infected cells. Activation of the NOD2/ NF-κB signaling axis is linked to the dysregulated overexpression of the PG remodeling enzyme AmiA and the subsequent cleavage and mislocalization of D-Ala-D-Ala analog. Inhibiting amiA transcriptional upregulation by CRISPR interference reduced pathogen recognition. We propose that nutritional immunity in general mediate abnormal expression of bacterial genes linked to pathogen-associated molecular patterns. Importance Limiting pathogen access to essential nutrients is the central tenet of nutritional immunity, with the outcome being severe starvation and eventual death of the pathogen. However, pathogen starvation induces several physiological changes prior to its death. They include errors in several biological processes, including metabolism and gene expression, which could lead to pathogen death. Here, we demonstrate that iron starvation of the clinically relevant human pathogen Chlamydia trachomatis significantly dysregulates the expression of a peptidoglycan remodeling amidase, AmiA to enhance chlamydial recognition by the host cell and the subsequent increased production of tumor necrosis factor and death of infected cells to the detriment of Chlamydia .

Abstract RNA-level detection is a critical approach for determining whether genetic variants affect splicing and for identifying potential pathogenic variants. However, tissues where disease-causing genes are expressed are often difficult to obtain in routine testing, while readily accessible tissues like blood do not express all disease-causing genes. CRISPR activation (CRISPRa) is a powerful technique for activating endogenous gene expression, and its application in peripheral blood mononuclear cells (PBMCs) offers an effective strategy for RNA analysis. In this study, we used CRISPRa to activate the expression of three disease-associated genes— FBN1 , F8 , and DMD —in PBMCs, achieving upregulation of target gene expression within 48 hours. In patient-derived cells, activation of FBN1 expression revealed that a deletion of exons 48–53 leads to a 708-bp in-frame deletion variant at the mRNA level. Notably, this CRISPRa-based method enabled the validation of splicing site variants within 3 days, significantly reducing turnaround time for clinical testing. Furthermore, we successfully activated 83 common disease-causing genes and established a single-guide RNA (sgRNA) library platform for activatable genes. This resource facilitates broader validation and screening of splicing site variants. Overall, this is the first study to demonstrate the use of CRISPRa for RNA-level variant detection in patient PBMCs. Our approach provides a novel approach for validating splicing site variants, including variants of unknown significance, as pathogenic in real clinical settings.

Severe loss of function variants in the splicing regulatory protein RBM10 are known to cause TARP syndrome, a rare X-linked recessive congenital syndrome. In recent years, individuals with milder phenotypes have been published, suggesting a broader phenotypic spectrum. We report 37 new individuals with RBM10 variants and compare to 34 published cases. We find that the phenotype can be described as an “RBM10-phenotypic spectrum” which can be further subdivided into two phenotypic groups, TARP syndrome (TARPS) and RBM10 Associated Intellectual Disability (RAID). Based on phenotype characterizations and functional studies, we describe a clear genotype-phenotype correlation. Splicing analysis of blood samples and CRISPR-edited cells representing different degrees of functional loss of RBM10 demonstrated a pattern of more exon inclusion in response to increased loss of RBM10 function. More inclusion was correlated with increasing phenotype severity. Functional studies of missense variants from the different phenotypic groups confirm this genotype-phenotype correlation and show that different molecular mechanisms can explain the underlying pathological alterations in RBM10 protein function. Interestingly, we show that some missense variants in the RNA binding, RRM2 domain of RBM10 alter RBM10 activity from splicing inhibition to stimulation, likely due to altered RNA binding characteristics. Graphical Abstract

Cocaine use disorder is highly comorbid in people with HIV and can accelerate infection, alter neuropathology, and exacerbate cognitive decline despite antiretroviral therapy (ART). Many of these effects are due to infection and dysregulation of CNS myeloid cells, especially microglia, which comprise a significant reservoir in this compartment. However, the precise mechanism(s) by which cocaine (Coc) enhances HIV infection in microglia are unclear, partly due to the lack of translationally relevant human microglial models suitable for mechanistic evaluation of Coc-mediated changes in viral dynamics. Canonically, Coc acts by blocking dopamine transporter activity, however, Coc has additional mechanisms beyond dopaminergic tone, involving the endoplasmic reticulum (ER) protein sigma-1, which has diverse cellular functions, including modulation of stress pathways such as the unfolded protein response (UPR). Viruses, including HIV, can exploit the UPR to amplify stress-induced protein production in host cells, enhancing viral replication. Therefore, we hypothesized that Coc-mediated activation of sigma-1 increases HIV infection of microglia via activation of the UPR. Using human-induced pluripotent stem cell microglia (iPSC-Mg), we evaluated changes in the percentage of infected iMg as well as p24Gag secretion using high-content imaging and AlphaLISA. Coc increased both p24 secretion and the number of infected iMg. The enhanced p24 secretion persisted despite ART, without the corresponding increase in percent p24, suggesting Coc augments the post-entry steps of viral replication. Inhibition of dopamine receptors did not diminish the impact of Coc, but pharmacological inhibition and CRISPR KO of sigma-1 blocked the effect. Independently, sigma-1 agonists increased p24 secretion, suggesting that Coc acts through sigma-1 rather than dopaminergic pathways. Single-cell RNA sequencing revealed distinct transcriptomic alterations in HIV+Coc-treated iPSC-Mg. Further genetic and proteomic validation confirmed activation of the IRE1-XBP1 branch of the UPR in the HIV+Coc condition, with increased XBP1 signaling and downstream cytokine (IL-4 and IL-7) secretion. The HIV+Coc condition also showed reduced expression of antiviral response genes and enhanced HIV transcriptional regulation genes. Immunofluorescence staining showed increased sigma-1 in p24-cells and reduced sigma-1 in p24+ cells in HIV+Coc cultures and revealed that HIV+Coc promotes sigma-1 movement to the ER. These findings suggest that Coc exploits sigma-1 signaling to modulate the UPR, enhancing viral replication and immune evasion. Sigma-1 emerges as a critical link between HIV-induced cellular stress and cocaine exposure, highlighting a shared molecular pathway that can be leveraged for the treatment of comorbid HIV neuropathogenesis, substance use disorder, and related neuropsychiatric disorders.

ABSTRACT As the largest organelle, the nucleus endures significant mechanical stresses over the cellular lifespan, and mechanostability, i.e. the ability to resist deformation, is critical for genome integrity and function. Here, we reveal that nuclear mechanostability is an emergent property arising from the clustering of satellite DNA repeats into nuclear condensates known as chromocenters. Targeted chromocenter disruption in Drosophila testes subjected to natural and artificial mechanical stress compromises nuclear mechanostability, leading to deformed nuclei, DNA damage, and chromosome breaks. Conversely, enhancing chromocenter coalescence through genetic means improves mechanostability. Molecular dynamics simulations suggest that chromocenters enable physically linked chromosomes to respond collectively, rather than individually, to mechanical challenge, and dissipate external forces over a larger nuclear surface. We propose that the satellite DNA-dependent mechanostability framework described here likely extends to other cells and tissues facing mechanical stress, and offers an explanation for the evolutionary success of these non-coding repeats across eukaryotes.

Cryptochromes (CRYs) are photolyase-like blue-light / ultraviolet-A (UV-A) receptors that regulate diverse aspects of plant growth. Maize ( Zea mays ), a major crop often grown under high UV-B radiation, possesses four copies of CRY. However, it remains unclear whether the multiple copies of CRY in maize have evolved to improve UV tolerance or to acquire new functions. In this study, CRISPR-Cas9-engineered Zmcry mutants were used to investigate the functions of four cryptochromes (ZmCRYs) in maize. The findings revealed that ZmCRYs play a redundant role in mediating blue light signaling and in inhibiting the elongation of the mesocotyl. The results also demonstrated that ZmCRYs mediated blue light-enhanced UV-B stress tolerance in Zea mays by upregulating the expression of genes involved in UV-B stress tolerance-related metabolites such as phenylpropanoid, flavonoid, and fatty acid biosynthesis. Furthermore, blue light was found to influence both the accumulation and composition of epidermal waxes, suggesting that blue light enhances epidermal wax accumulation for UV-B stress tolerance. Additionally, it was discovered that ZmCRY1 directly interacted with GLOSSY2 (GL2) in a blue light dependent manner to mediate blue light promoted C32 aldehyde accumulation, shedding new light on the enigma of aldehyde-forming. These results highlight the critical roles of ZmCRY1s in mediating blue light regulated epidermal wax biosynthesis and UV-B tolerance in Zea mays .

Genome wide association study (GWAS) reports substantially outpace subsequent functional characterization. Pinpointing the causal effector gene(s) at GWAS loci remains challenging given the non-coding genomic residency of >98% of these signals. We previously implicated effector genes at GWAS loci for the complex and polygenic disorder of human insomnia using a high-resolution cell-specific, chromatin capture-based variant-to-gene mapping protocol, paired with sleep phenotyping in Drosophila . In this study, we leveraged a diurnal vertebrate model with higher genomic conservation, namely zebrafish, to screen our six highest confidence candidate genes and identify those whose loss-of-function impaired sleep characteristics related to human insomnia-like behaviors. Of these genes, we observed that CRISPR-mediated deletion of the zebrafish ortholog of MEIS1 produced nighttime specific sleep fragmentation and increased latency to sleep, pointing to a conserved role for MEIS1 in sleep maintenance. Comparing our human cell-based chromatin accessibility and contact maps with publicly available zebrafish spatial genomic data revealed highly conserved genomic architecture harboring the insomnia GWAS variant of interest. Notably, this genomic conservation was selective for the zebrafish ortholog which contributed to the sleep phenotype, meis1b, while the duplicated ohnolog meis1a proved dispensable. Motivated by this, we characterized the spatio-temporal expression of meis1b in zebrafish, showing it is comparable to human with respect to cerebellar granule progenitors. Ultimately, we found that loss of meis1b impairs cerebellar development. Together, our work provides a powerful model for screening human disorder risk genes for sleep fragmentation using a tractable vertebrate and supports a conserved cerebellar role for MEIS1 in sleep disturbance.

G protein-coupled receptors (GPCRs) that couple to the Gαq signaling pathway control diverse physiological processes, yet the full complement of cellular regulators for this pathway remains unknown. Here, we report the first genome-wide CRISPR knockout screen targeting a Gαq-coupled GPCR signaling cascade. Using a Drosophila model of adipokinetic hormone receptor (AkhR) signaling, we identified CG34449 (Zdhhc8) , encoding a palmitoyl acyltransferase and its adapter protein CG5447, as a top hit required for robust Gαq-mediated GPCR signaling. We show that Zdhhc8 enhances GPCR signaling through palmitoylation of Gαq, which promotes its membrane localization and function. Loss of Zdhhc8 markedly reduces palmitoylation of Gaq resulting in attenuation of AkhR/Gαq signaling and a reduction in receptor stability. Mechanistically, Zdhhc8 is necessary for palmitoylation of Gαq. These findings uncover Zdhhc8-dependent Gαq palmitoylation as a pivotal regulatory mechanism in GPCR signal transduction and highlight palmitoyl transferase as potential modulators of GPCR pathways.

Abstract 2.1 Background Adipogenesis is a highly organised series of events that facilitates the healthy expansion of adipose tissue, beginning during embryogenesis and continuing throughout life. White adipogenesis protects against lipotoxicity, influencing insulin resistance and obesity-related comorbidities. Brown adipogenesis enhances energy expenditure, thereby counteracting weight gain, lipotoxicity and insulin resistance. Recently, there has been a significant increase in interest regarding adipocyte differentiation, mainly focusing on the interplay between microRNAs (miRNAs) and the transcriptional cascade that governs adipogenesis and metabolic dysfunction. This study aimed to identify miRNAs regulating white and brown adipocyte differentiation and define miRNA action in a stem cell model of adipogenesis. 2.2 Methods Small RNAseq analysis of primary mouse brown and white adipocytes (WAs) identified miR-10b to be upregulated in mature brown adipocytes (BAs). We generated two model systems: 1) immortalized brown pre-adipocytes treated with miRNA inhibitors and 2) CRISPR/Cas9 KO of miR-10b in E14 mouse embryonic stem cells (mESCs). Both cell models were differentiated into mature adipocytes. To unravel the pathways that are affected by miR-10b depletion, a transcriptomic analysis was performed at key time points. 2.3 Results Both cell models showed that miR-10b-5p depletion severely impaired differentiation into mature adipocytes, as indicated by a lack of lipid droplet formation and reduced adipogenic gene expression. Gene expression analysis supports that miR-10b-5p directs embryonic stem (ES) cells towards the mesoderm lineage, promoting commitment to pre-adipocytes by downregulating Gata6 and its downstream target Bmp2. This mechanism appears to be unaffected in BAs. Our study demonstrated that miR-10b-5p regulates the later stages of adipogenesis, at least in part, by downregulating Tub, a direct target of miR-10b-5p. We also confirmed that miR-10b-5p alleviated the halted differentiation phenotypes of adipocytes by supressing the G Protein Signalling pathway mediated by Tubby. 2.4 Conclusions These results evidence that miR-10b inhibition plays a dynamic role in adipocyte biology, as its inhibitory effects manifest differently during the stem cell preadipocyte proliferation state and during the maturation phase of adipocytes. Collectively, our study demonstrated that miR-10b-5p may represent a new potential therapeutic target for lipodystrophy and obesity.

Abstract Background DOT1L, a histone H3 lysine 79 (H3K79) methyltransferase, is a potential therapeutic target in various malignancies. In the present study, we aimed to clarify the antitumor effect of DOT1L inhibition in breast cancer. Methods Estrogen receptor (ER)-positive/HER2-negative breast cancer cells (MCF7) and ER-negative/HER2-positive cells (SKBR3) were treated with a DOT1L inhibitor (SGC0942, EPZ-5676), after which colony formation assays, cell cycle assays, flow cytometry, gene expression microarray analysis, chromatin immunoprecipitation sequencing (ChIP-seq) and single-cell Assay for Transposase-Accessible Chromatin sequencing (scATAC-seq) were performed. Genetic ablation of STING was performed using the CRISPR/Cas9 system. Results Treatment with a DOT1L inhibitor suppressed proliferation and induced cell cycle arrest and apoptosis in both ER-positive/HER2-negative and ER-negative/HER2-positive cells. Transcriptome and epigenome analysis revealed that DOT1L inhibition activated transcription of a number of interferon (IFN)-related genes (IRGs) in breast cancer cells. We also found that DOT1L inhibition upregulated type I and type III IFNs and cell surface human leukocyte antigen (HLA) class I expression. Notably, DOT1L inhibition induced DNA damage and upregulated levels of cytoplasmic DNA in breast cancer cells. CRISPR/Cas9-mediated knockout of STING in breast cancer cells significantly suppressed the IFN signaling activated by DOT1L inhibition and attenuated the antitumor effects. Moreover, scATAC-seq analysis revealed that DOT1L inhibition suppressed expression of ERBB2 in HER2-positive breast cancer cells. Conclusions These findings suggest that the anti-breast cancer cell effects of DOT1L inhibition are mediated by multiple mechanisms, including activation of innate immune signaling.

Membrane protection against oxidative insults is achieved by the concerted action of glutathione peroxidase 4 (GPX4) and endogenous lipophilic antioxidants such as ubiquinone and vitamin E. Deficiencies in these protective systems lead to an increased propensity to phospholipid peroxidation and ferroptosis. More recently, ferroptosis suppressor protein 1 (FSP1) was identified as a critical ferroptosis inhibitor acting via regeneration of membrane-embedded antioxidants. Yet, regulators of FSP1 are largely uncharacterised, and their identification is essential for understanding the mechanisms buffering phospholipid peroxidation and ferroptosis. Here, we conducted a focused CRISPR-Cas9 screen to uncover factors influencing FSP1 function, identifying riboflavin (vitamin B₂) as a new modulator of ferroptosis sensitivity. We demonstrate that riboflavin, unlike other vitamins that act as radical-trapping antioxidants, supports FSP1 stability and the recycling of lipid-soluble antioxidants, thereby mitigating phospholipid peroxidation. Furthermore, we show that the riboflavin antimetabolite roseoflavin markedly impairs FSP1 function and sensitises cancer cells to ferroptosis. Thus, we uncover a direct and actionable role for riboflavin in maintaining membrane integrity by promoting membrane tolerance to lipid peroxidation. Our findings provide a rational strategy to modulate the FSP1-antioxidant recycling pathway and underscore the therapeutic potential of targeting riboflavin metabolism, with implications for understanding the interaction of nutrients and their contributions to a cell’s antioxidant capacity.

ABSTRACT Ferroptosis, a regulated form of cell death driven by excessive lipid peroxidation, has emerged as a promising therapeutic target in cancer. Ferroptosis suppressor protein 1 (FSP1) is a critical regulator of ferroptosis resistance, yet the mechanisms controlling its expression and stability remain mostly unexplored. To uncover regulators of FSP1 abundance, we conducted CRISPR-Cas9 screens utilizing a genome-edited, dual-fluorescent FSP1 reporter cell line, identifying both transcriptional and post-translational mechanisms that determine FSP1 levels. Notably, we identified riboflavin kinase (RFK) and FAD synthase (FLAD1), enzymes which are essential for synthesizing flavin adenine dinucleotide (FAD) from vitamin B2, as key contributors to FSP1 stability. Biochemical and cellular analyses revealed that FAD binding is critical for FSP1 activity. FAD deficiency, and mutations blocking FSP1-FAD binding, triggered FSP1 degradation via a ubiquitin-proteasome pathway that involves the E3 ligase RNF8. Unlike other vitamins that inhibit ferroptosis by scavenging radicals, vitamin B2 supports ferroptosis resistance through FAD cofactor binding, ensuring proper FSP1 stability and function. This study provides a rich resource detailing mechanisms that regulate FSP1 abundance and highlights a novel connection between vitamin B2 metabolism and ferroptosis resistance with implications for therapeutic strategies targeting FSP1 in cancer.

ABSTRACT Escherichia coli ( E. coli ) is a common bacterium in the human gut and an important cause of intestinal and extraintestinal infections. Some E. coli sequence types (ST) are associated with high pathogenicity. The Extraintestinal Pathogenic E. coli (ExPEC) ST131 is a globally distributed multidrug-resistant human pathogen associated with urinary tract and bloodstream infections. Antibiotic-resistant infections often lead to antibiotic treatment failure, underscoring the need of developing alternative treatments. The highly selective antimicrobial potential of CRISPR-Cas9 has been demonstrated in a range of model organisms. However, the effectiveness of CRISPR-Cas9 in combating ST131-associated infections and the consequences of CRISPR-Cas9 treatment, such as the emergence of escapers, remains unclear. Here, we investigated the antimicrobial activity of CRISPR-Cas9 against ST131 and assessed the frequency and genetic basis of escape. We conjugatively delivered CRISPR-Cas9 to ST131 isolates which carried cefotaxime-resistance-encoding target gene bla CTX-M-15 in the chromosome and characterized escape subpopulations. Two main types of escapers emerged: bla CTX-M-15 -positive escapers carried dysfunctional CRISPR-Cas9 systems and arose at a ∼10 −5 frequency. Instead, bla CTX-M-15 -negative escapers presented chromosomal deletions involving bla CTX-M-15 loss. The frequency of bla CTX-M-15 loss depended on the bla CTX-M-15 genetic context. Specifically, bla CTX-M-15 -negative escapers emerged at low frequency (∼10 −5 ) in isolates where bla CTX-M-15 was located downstream of insertion sequence (IS) IS Ecp1 , while escapers emerged with high frequency (∼10 −3 ) in isolates where bla CTX-M-15 was flanked by IS 26 . This work emphasizes how the genetic context of target genes can drive the outcome of CRISPR-Cas9 tools, where the presence of IS 26 may drive increased frequencies of escape. IMPORTANCE In the past decade CRISPR-Cas9 has emerged as an efficient antimicrobial tool capable of selective elimination of targeted bacteria. Even though it has been well described that bacteria can evolve to escape targeting by CRISPR-Cas9, the mechanisms of bacterial escape and their consequences remain largely elusive. In this study, we demonstrate the antimicrobial efficacy of CRISPR-Cas9 against natural isolates of Escherichia coli ST131, a clinically relevant pathogen, and elucidate the mechanism of escape from antimicrobial activity. We identify two distinct mechanisms of escape, which involve either dysfunctional CRISPR-Cas9 activity, or loss of the target gene ( bla CTX-M-15 ), with the latter occurring at frequencies that depend on the genetic context of the target gene. These findings provide important insights into the frequency and mechanisms of bacterial escape from CRISPR-Cas9-based antimicrobials and offer a foundation for the development of more effective treatments.

LMX1B, a LIM-homeodomain family transcription factor, plays critical roles in the development of multiple tissues, including limbs, eyes, kidneys, brain, and spinal cord. Mutations in the human LMX1B gene cause the rare autosomal-dominant disorder, Nail-patella syndrome which affects development of limbs, eyes, brain, and kidneys. In zebrafish, lmx1b has two paralogues: lmx1ba and lmx1bb. While lmx1b morpholino data exists, stable mutants were previously lacking. Here we describe the characterisation of lmx1b stable mutant lines, with a focus on development of tissues which are affected in Nail-patella syndrome. We demonstrate that the lmx1b paralogues have divergent developmental roles in zebrafish, with lmx1ba affecting skeletal and neuronal development, and lmx1bb affecting renal development. The double mutant, representing loss of both paralogues (lmx1b dKO) showed a stronger phenotype which included additional defects to trunk muscle patterning, and a failure to fully inflate the notochord leading to a dramatic reduction in body length. Overall, these mutant lines demonstrate the utility of zebrafish for modelling Nail-patella syndrome and describe a previously undescribed role for lmx1b in notochord cell inflation.

ABSTRACT Recent breakdowns in the supply chains of antiretroviral therapy (ART) to lower income countries, where they are needed most, underpin the pressing need for an accessible and scalable HIV cure that would allow ART-free control of the virus. The ‘shock-and-kill’ cure concept shows promise in its ability to reactivate HIV-1 transcription in latently infected cells in vivo in people living with HIV-1 but has failed to result in a meaningful reduction of the size of the reactivatable viral reservoir. We therefore hypothesised that the efficiency of reversal of transcriptional quiescence, and the resulting HIV-1 antigen expression and presentation, may be insufficient to achieve full immunological visibility of HIV-1-infected cells. To gain a deeper understanding of potential LRA-specific shortcomings, we developed a novel model of HIV-1 latency - Jurkat E6.1 subclonal cell lines, each harbouring a single, full-length, NL4.3-based provirus with a GFP OPT reporter inserted into the V5 loop of the viral Env protein. Using this model, we quantified HIV-1 reactivation, as well as expression and surface presentation of Env after treatment with a panel of known latency reversal agents (LRAs) and their synergistically-acting combinations. HIV-1 reactivation with the PKC agonist Bryostatin-1 limited the cell-surface presentation of viral Env, a phenomenon we found being linked to Bryostatin-1’s ability to induce the expression of the restriction factor GBP5 in T-cell lines and primary CD4+ T-cells, and induced a cellular state which was resistant to apoptosis. A combined treatment of Bryostatin-1 and the BET inhibitor (iBET) JQ1 resulted in synergistic HIV-1 reactivation and boosted levels of cell-surface Env, but failed to reduce resistance to apoptosis. In contrast, the combination of the SMAC mimetic (SMACm) AZD5582 and JQ1 markedly boosted cell-surface Env levels, without co-induction of GBP5, T-cell activation or apoptotic resistance. Crucially, HIV-1 Nef was found to be a potent antagonist of the cytotoxic killing of reactivating cells, most likely by its ability to inhibit apoptosis. Nef knockout, however, when paired with the AZD/JQ1 combination, displayed highest potency in inducing immune-mediated elimination of latently infected T-cells and presents a promising new approach for HIV cure programs.

The identification of novel antimalarials with activity against both the liver and blood stages of the parasite lifecycle would have the dual benefit of prophylactic and curative potential. However, one challenge of leveraging chemical hits from phenotypic screens is subsequent target identification. Here, we use in vitro evolution of resistance to investigate nine compounds from the Tres Cantos Antimalarial Set (TCAMS) with dual liver and asexual blood stage activity. We succeeded in eliciting resistance to four compounds, yielding mutations in acetyl CoA synthetase (AcAS), cytoplasmic isoleucine tRNA synthetase (cIRS), and protein kinase G (PKG) respectively. Using a combination of CRISPR editing and in vitro activity assays with recombinant proteins, we validate these as targets for TCMDC-125075 (AcAS), TCMDC-124602 (cIRS), and TCMDC-141334 and TCDMC-140674 (PKG). Notably, for the latter two compounds, we obtained a T618I mutation in the gatekeeper residue of PKG, consistent with direct interaction with the active site, which we modelled with molecular docking. Finally, we performed cross-resistance evaluation of the remaining five resistance-refractory compounds using the Antimalarial Resistome Barcode sequencing assay (AReBar), which examined a pool of 52 barcoded lines with mutations covering >30 common modes of action. None of the five compounds where in vitro evolution of resistance was not successful yielded validated hits using AReBar, indicating they likely act via novel mechanisms and may be candidates for further exploration.

Here we present pCASKD, a single-plasmid system for scarless chromosomal editing in Escherichia coli . Our plasmid pCASKD integrates CRISPR-Cas9-mediated counterselection, Lambda-Red recombineering, and temperature-sensitive plasmid curing into a 12 Kb vector to enable kilobase-scale insertions and deletions using a linear dsDNA donor and homologous recombination. Using the flagellar stator motAB locus as a model, we demonstrate that pCASKD enables efficient knock-in and knock-out edits with lower donor DNA input and reduced false positives compared with its parent, multi-plasmid system, No-SCAR. Using a single plasmid reduces transformation steps, accelerates screening, and increases the frequency of correctly edited clones. The protocol can be completed in five days, with potential for further optimization, offering a compact and efficient alternative for microbial genome engineering.

ABSTRACT Site-directed RNA editing, especially RNA base editing, allows for specific manipulation of RNA sequences, making it a useful approach for the correction of pathogenic mutations. Correction of RNA transcripts allows therapeutic gene editing in a safe and reversible manner and avoids permanent alterations in the genome. RNA-targeting CRISPR-Cas nucleases (e.g., CRISPR-Cas13) enable delivery within a single adeno-associated virus (AAV) vector for RNA base editing, making the approach clinically feasible. Here, we used the inactive CRISPR-Cas13bt3 (also known as Cas13X.1) fused to the ADAR2 deaminase domain (ADAR2DD) for targeted correction of inherited retinal disease (IRD) mutations. First, we show in vitro that dCas13bt3-ADAR2DD can efficiently correct a pathogenic nonsense mutation (c.130C>T [p.R44X]) found in the mouse Rpe65 gene and recover protein expression in retinal pigment epithelium cells (RPEs). Across clinically reported RPE65 mutations, we observed editing efficiencies ranging from 0% to 60%. In the Rpe65 -deficient mouse model of retinal degeneration (rd12), we observed that RNA base editing can recover Rpe65 expression in RPEs and rescue retinal function with no observable adverse effects. We further employed our RNA base editor against the large USH2A gene to assess the promise of RNA base editing for addressing untreatable IRDs caused by genes too large for AAV gene delivery. Against the human USH2A in vitro , we observed up to 60% on-target efficiency. We further found that gRNA mismatches, domain-inlaid ADAR2DD design and nucleocytoplasmic shuttling of the RNA base editor optimised on-target and bystander editing for a highly precise base editor. Against the mouse Ush2a in vitro , we similarly observed up to 60% on-target editing in mammalian cells, while in the Ush2a W3947X mice, we observed ∼12% on-target editing, with no impact on retinal structure or function, or transcriptome-wide editing. Overall, our findings demonstrate dCas13bt3-ADAR2DD as a potent tool for gene therapy against IRDs, addressing a significant unmet clinical need in ophthalmology.

Some phages have evolved the ability to cooperate to evade the immunity triggered by their bacterial host. A first exposure to the phage may weaken the host defences and allow later infections to be successful. Because this cooperation requires sequential infections, the phage can invade the host population only if its initial density is sufficiently high in a well-mixed environment. However, most natural bacterial populations are spatially structured. Could spatial structure create more favourable conditions for viral spread? Here we study the effect of spatial structure on the dynamics of cooperative anti-CRISPR (Acr) phages spreading in a population of CRISPR-Cas resistant Pseudomonas aeruginosa bacteria. We show experimentally that spatial structure does not always promote the spread of Acr-phages. In particular, the effect of spatial structure is modulated by the efficacy of the bacterial host’s CRISPR-Cas resistance and by the efficacy of the phage Acr protein. These results are discussed in the light of a mathematical model we developed to describe the spread of the phage. The model allows us to understand the ambivalent effects of spatial structure via its effects on the reproduction and on the persistence of the phage. More generally, we find that spatial structure can have opposite effects on the epidemiological dynamics of the phage, depending on the properties of the Acr protein encoded by the phage. This joint experimental and theoretical work yields a deeper understanding of the spatial dynamics of cooperative strategies in phages.

Understanding the gene regulatory mechanisms underlying brain function is crucial for advancing knowledge of the genetic basis of neurologic diseases. Cis-regulatory elements (CREs) play a pivotal role in gene regulation, and their evolutionary conservation can offer valuable insights. Importantly, the function and evolution of CREs are affected not only by primary sequence, but also by the cis - and trans -regulatory context. However, comparative functional analyses across species have been limited, leaving how these regulatory landscapes evolve in the brain largely unresolved. Here, we generated single-nucleus multiomic (snRNA- and snATAC-seq) data from cortex tissue across nine mammalian species and identified candidate CREs (cCREs) in a cell type–specific manner. We developed a multidimensional framework of conservation to assess sites of shared function that integrates sequence, chromatin accessibility, and enhancer–gene associations. Using massively parallel reporter assays (MPRA) in human neural progenitor cells and neurons, we measured activity of cCREs including both conserved and human-specific regions. CRISPR interference validated conserved enhancer function, including at neurodevelopmentally important genes like FAM181B . Motif enrichment identified transcription factors distinguishing conserved versus recent cCREs. Linkage disequilibrium score regression indicated that both conserved and human-specific cCREs were enriched for neuropsychiatric GWAS risk, while neurodegenerative risk was confined to conserved elements. Our findings define functional dimensions of enhancer conservation and demonstrate how regulatory evolution shapes human brain biology and disease susceptibility.

Genetic interactions are typically studied by looking at the phenotype that results from disruption of pairs of genes, as well as from higher order combinations of perturbations. Systematically interrogating all pairwise combinations provides insights into how genes are organized into pathways and complexes to sustain cellular homeostasis and how interacting genes respond to stressors and external signals. Genetic interactions have been studied extensively in yeast, due, in part, to the availability of a systematic collection of gene knockouts, and the development of Synthetic Genetic Array (SGA) technology. In contrast, such approaches are more challenging in human cells and therefore comparable data for human cells is scarce. This study introduces an innovative approach to functionally characterize genetic interactions in human cells through CRISPR/Cas9 screens using a pooled genome-wide knockout library in NALM6 cells. By combining a single guide RNA (sgRNA) targeting the gene of interest (aka the query) in cells already infected with an inducible genome-wide sgRNA pool, it is possible to achieve near saturation of genome-wide double knockouts. We conducted 26 of these screens, which we term “gene by genome-wide” knockout screens. This approach can be rapidly performed, in part, because it bypasses the need to generate genotyped isogenic knockout clones. Data from these screens identified both expected and novel synthetic lethal and synthetic rescue interactions, demonstrating that this strategy is effective for large-scale genetic research in human cells. Additionally, we show that these GBGW screens can be combined with chemical perturbation to reveal new synthetic interactions that are not apparent without drug treatment. Finally, we show that cDNA overexpression can be incorporated with genome-wide knockouts to systematically explore gain-of-function scenarios. The complete dataset is accessible on the ChemoGenix website (URL: https://chemogenix.iric.ca ).

Human papillomavirus (HPV) is a major etiological factor in cervical, anal, and oro-pharyngeal cancers. Although prophylactic vaccines have substantially reduced infec-tion rates, effective therapeutic options for established HPV-associated malignancies remain limited. This review provides an up-to-date overview of emerging strategies to treat HPV-driven tumors. Key approaches include immune checkpoint inhibitors, therapeutic vaccines such as VGX‑3100 and PRGN‑2012, and gene-editing tools like CRISPR/Cas9. Epigenetic drugs, particularly histone deacetylase inhibitors, show promise in reactivating silenced tumor suppressor genes and enhancing antitumor immunity. In addition, natural bioactive compounds and plant-derived molecules are being explored as complementary anti-HPV agents, while drug repurposing and combination therapies offer cost-effective opportunities to broaden treatment options. We also highlight the role of patient-derived organoid models as powerful platforms for personalized drug screening and functional assessment. By integrating these therapeutic innovations with precision oncology approaches, this review outlines a multi-dimensional framework aimed at improving clinical outcomes and quality of life for patients with HPV-associated cancers.

Terpenoids, vital pharmaceutical compounds, face production challenges due to low yields in native plants and ecological concerns. This review synthesizes recent advances in metabolic engineering strategies implemented across three complementary platforms: native medicinal plants, microbial systems, and heterologous plant hosts. We elucidate how the "Genomic Insights to Biotechnological Applications" paradigm, empowered by multi-omics technologies such as genomics, transcriptomics, metabolomics, etc., drives research advancements. These technologies facilitate the identification of key biosynthetic genes and regulatory networks. CRISPR-based tools, enzyme engineering, and subcellular targeting are highlighted as transformative strategies. Significant yield improvements have been demonstrated, with artemisinin and paclitaxel precursors showing considerable increases in production through strategic co-expression and optimization techniques. Persistent challenges such as metabolic flux balancing, cytotoxicity, and scale-up economics are discussed alongside emerging solutions including machine learning and photoautotrophic chassis. We conclude by outlining a roadmap for industrial translation that emphasizes the critical integration of systems biology and synthetic biology approaches to accelerate the transition of terpenoid biomanufacturing from discovery to commercial scale.

Genome editing technologies including CRISPR/Cas9, TALENs, and ZFNs offer a unique opportunity to eradicate HPV by directly disrupting its oncogenes E6 and E7, thereby restoring tumour‑suppressor pathways. In this review, we provide a comprehensive overview of recent advances in HPV‑targeted genome editing, summarising key preclinical findings that demonstrate tumour regression and apoptosis in HPV‑positive models, as well as the first‑in‑human clinical trials assessing safety and feasibility of local CRISPR‑based therapies. We also compare the relative strengths and limitations of each editing platform, discuss delivery strategies, and highlight their potential integration with immunotherapy and standard cancer treatments. While genome editing shows unprecedented precision and durability in targeting viral oncogenes, challenges such as efficient delivery, minimising off‑target effects, and navigating regulatory and ethical considerations remain. Continued optimisation of high‑fidelity nucleases, tissue‑specific delivery vehicles, and personalised guide design will be essential to translate these promising approaches into routine oncology practice. Genome editing thus represents a paradigm shift in HPV therapy, with the potential to transform management of both persistent infections and established cancers.

DNASE1L3 is a key endonuclease, essential for proper fragmentation and clearance of cell-free DNA (cfDNA). The p.R206C common variant impairs DNASE1L3 secretion and activity, causing aberrant cfDNA fragmentation and therefore affecting liquid biopsy-based screening and diagnostics. Existing studies on DNASE1L3 relied on resource-intensive murine models or plasmid-based overexpression, which do not accurately represent native expression. To address this, we developed an isogenic HEK293T cell line model by using CRISPR Prime Editing for endogenous expression of DNASE1L3 R206C . We analyzed the cfDNA composition directly from conditioned culture medium and found that fragment size distributions in mutant cells mimics the hypofragmented profiles previously observed in plasma samples from p.R206C carriers. We also showed that in vitro treatment of hypofragmented cfDNA with recombinant wildtype DNASE1L3 could enrich for mononucleosomal fragments, with fragment end-motifs characteristic of DNASE1L3 cleavage activity. This could open avenues for DNASE1L3 as a candidate pre-treatment agent to improve the accuracy and efficiency of cfDNA sequencing-based diagnostics in hypofragmented liquid biopsies. These findings demonstrate that our isogenic cell line model provides a controlled system to study cfDNA fragmentation biology and DNASE1L3 function.