The isomerohydrolase RPE65 is a critical element of the visual cycle, the series of enzymatic reactions by which the chromophore of the visual pigments is regenerated following light exposure. In humans, mutations in the rpe65 gene cause a severe form of blindness called Leber’s congenital amaurosis. Studies of RPE65 -/- mice have shown dramatic depletion of 11- cis-retinal in the retina, resulting in a slow retinal degeneration. However, a number of studies suggest that RPE65 may not be necessary for the regeneration of photopigment in all photoreceptor types. Using CRISPR/Cas9 technology, we previously generated RPE65 knockout Xenopus laevis in order to test the involvement of rhodopsin chromophore in the cell death mechanisms associated with rhodopsin mutations and rhodopsin quality control. Here we further characterize the effects of RPE65 knockout in these animals, and show their rod photoreceptors have shortened outer segments that lack detectable rhodopsin photopigment. However, there is no progressive degeneration of rods or cones. Via electroretinography we found greatly reduced but significant responses to light under scotopic and photopic conditions. We also found reduced behavioral sensitivity to light, while light-induced melanophore dispersion was unaffected. RPE65 knockout X. laevis may be a useful system for examining RPE65-independent photosensation mechanisms in vertebrates.

Most microbial taxa on Earth remain uncultivated, limiting our ability to study their physiology, ecology, and roles in environmental processes. Although metagenome-assembled genomes (MAGs) have expanded access to uncultured phylogenetic diversity, the functional basis for culturability remains poorly understood. Here, we analyze the 52,515 MAGs from the Genomes from Earth’s Microbiomes (GEM) catalog to test two hypotheses: 1) genomes from uncultured microbes encode more functionally novel genes than those from cultured taxa, and 2) specific genomic features are systematically associated with culturability across phyla. To assess functional novelty, we aligned predicted proteins to SwissProt and measured sequence dissimilarity to the nearest curated homolog. We find that uncultured MAGs, particularly among Archaea, harbor substantially more divergent proteins. To identify genomic traits predictive of culturability, we combined pathway-level enrichment with LASSO regression and permutation-based feature importance. Cultured MAGs were consistently enriched in Clusters of Orthologous Groups (COG) pathways related to vitamin and cofactor biosynthesis (e.g., thiamine, folate, B12), energy metabolism (e.g., TCA cycle), and CRISPR-Cas systems—functions often depleted in uncultured counterparts. LASSO models identified a subset of these pathways as strong predictors of cultured status even in poorly sampled phyla, suggesting conserved genomic signatures of culturability. In contrast, pathways such as purine biosynthesis and NADH dehydrogenase were associated with uncultured lineages, highlighting potential barriers to cultivation. These results 1) demonstrate the great functional novelty of uncultured microbes, potentially offering unprecedented opportunities for discoveries of novel function, and 2) identify metabolic traits associated with culturability to inform future cultivation strategies. Importance The vast majority of microbes are uncultured, which means they have never been characterized under laboratory conditions. We showed that genomic sequences of uncultured microbes have less similarity to characterized proteins compared to cultured microbes, revealing that there may be fundamental biological reasons why they are not cultured. We also showed that certain metabolic pathways, such as those related to vitamin and cofactor biosynthesis, can predict the ability of microbes to grow under laboratory conditions, and these pathways are abundant in highly cultured phyla, indicating how metabolic pathways can influence cultivation strategies.

Leishmania parasites cause a spectrum of diseases known as leishmaniases and must acquire nutrients like iron while surviving host defenses. Aquaglyceroporin 1 (AQP1) is a membrane channel that, in L. major , localizes to the flagellum and mediates antimony uptake and cell-volume regulation. Here, we show that in L. amazonensis AQP1 is instead targeted to glycosomes and that its expression is modulated by iron availability. A CRISPR-Cas9–mediated knockout of AQP1 in L. amazonensis revealed its multifunctional importance. AQP1-null promastigotes displayed a significant growth defect, particularly under iron-depleted conditions, and were impaired in regulating cell volume under osmotic stress. The mutant parasites contained approximately 50% less intracellular iron than wild-type cells and showed an increase in total superoxide dismutase activity, underscoring a role for AQP1 in iron homeostasis and oxidative stress management. AQP1 deletion also markedly reduced virulence in murine macrophages and in infected mice. Strikingly, loss of AQP1 increased resistance to trivalent antimony (Sb III ), a first-line antileishmanial drug. AQP1-knockout promastigotes showed a 70% increase in Sb III EC 50 and accumulated more Sb intracellularly than wild-type, suggesting an altered antimony handling. Altogether, L. amazonensis AQP1 is a glycosomal protein that links iron metabolism, osmoregulation, and antimony susceptibility. Its glycosomal targeting and multifaceted roles differ from those of AQP1 orthologs in other Leishmania species. These findings suggest the existence of additional antimony uptake mechanisms beyond AQP1, with implications for understanding drug resistance. Author Summary Leishmaniases are neglected tropical diseases caused by parasites that survive and multiply inside vertebrates’ cells. These parasites rely on hosts’ nutrients like iron and must resist both host defenses and treatment with toxic drugs such as antimony. We studied a protein called Aquaglyceroporin 1 (AQP1) in Leishmania amazonensis , a species that causes skin lesions in South America. Unlike related species, where AQP1 is found on the parasite’s surface, we discovered that in L. amazonensis AQP1 is located in an internal organelle called glycosome. By deleting this protein from the parasite, we found that it plays a crucial role in iron balance, sensitivity to antimony drugs, and the parasite’s ability to cause disease. Unexpectedly, parasites without AQP1 were more resistant to antimony but still accumulated high levels of the drug, suggesting that Leishmania has other ways of taking up antimony. Our findings challenge the assumption that all Leishmania species use the same strategies to survive, and highlight the need to understand species-specific differences when designing treatments or analyzing parasite biology.

SUMMARY Metastasis is an emergent continuum, driven by evolving reciprocal adaptations between continuously disseminating tumor cells (DTCs) and the specialized metastatic niches of distant organs. The interplay between intrinsic and niche-driven mechanisms that enables DTCs to survive and home to distant organs remains incompletely understood. Here, using MetTag, a single-cell barcoding and transcriptome profiling approach with time-stamped batch identifiers (BC.IDs), we mapped temporal, clonal dynamics of DTCs and the immune cell landscape across ovarian cancer metastatic niches. Deep sequencing of barcodes revealed preferred enrichment of early-disseminated clones across metastatic niches. Mechanistically, single-cell RNA sequencing (scRNA-seq) coupled with velocity analyses in ascites and metastasis-bearing omenta uncovered an emergent, distinct interferon-gamma (IFNγ) centric transcriptional trajectory, enriched among early seeding clones. Moreover, in vivo CRISPR/Cas9 screening of metastatic niche-specific signatures demonstrated that genes belonging to the ascites IFNγ signature, including Marco , Gbp2b, and Slfn1, are functionally important for peritoneal metastasis. Knockout of IFNγ receptor 1 ( Ifngr1 ) in tumor cells significantly reduced metastatic burden and extended survival, underscoring the importance of tumor cell intrinsic IFNγ signaling in ovarian cancer metastasis. Furthermore, we identified that the tumor intrinsic IFNγ response and ascites-derived tumor-associated macrophages (TAMs) protect cancer cells from anoikis-mediated death within the IFNγ-rich ascites environment. Our study resolves temporal dynamics of disseminating tumor cells and highlights an ascites-driven, IFNγ program as a necessary pro-metastatic adaptation in the ovarian metastasis cascade. Graphical abstract

ABSTRACT Germline pathogenic BRCA1 variants predispose women to breast and ovarian cancer. Despite accumulation of functional evidence for variants in BRCA1 , over half of reported single-nucleotide variants (SNVs) lack a definitive clinical interpretation. Furthermore, the extent to which variant effects are consistent across cell types remains largely unexplored. Here, we performed saturation genome editing (SGE) of BRCA1 in HAP1 cells to score 4,113 variants not previously assayed. Additionally, we developed a new SGE assay in human mammary epithelial cells (HMECs), allowing effects of variants to be compared across cell lines, drug treatments, and genetic backgrounds. We identify 538 variants impacting function via diverse mechanisms, including impairment of the BRCA1–PALB2 interaction and disruption of splicing, transcription, and translation. Function scores from experiments in HAP1 discriminate known pathogenic and benign variants with near-perfect accuracy. Intriguingly, however, nearly half of variants impacting function in HAP1 were found to be neutral when assayed in HMECs. We show that discordantly scored variants are hypomorphic and confer intermediate cancer risk. These results will be highly valuable for clinical interpretation of BRCA1 variants. Moreover, this work illustrates how revealing context-specific variant effects across cell types can enable more accurate resolution of disease risk.

ABSTRACT While NLRP3 has been extensively studied in myeloid cells, its existence and regulation in epithelial cells, including keratinocytes, are unclear. In fact, whether human keratinocytes express a functional NLRP3 inflammasome at all remains a matter of debate in the inflammasome field. Here, we provide additional evidence that NLRP3 is repressed in human keratinocytes cultured under non-inflammatory conditions but can be sharply induced by interferon-γ (IFNγ)—but not lipopolysaccharide (LPS). In this IFNγ-primed state, not all established NLRP3 activators are specific to NLRP3. We report that nigericin-driven keratinocyte pyroptosis occurs via both NLRP1 and NLRP3, whereas Staphylococcus aureus α-hemolysin (Hla) exclusively and nonredundantly activates NLRP3, even though both require K+ efflux. Furthermore, in the presence of T cells, certain virulent S. aureus strains can cause NLRP3-dependent pyroptotic death in keratinocytes in vitro through the cooperative actions of superantigens (SAgs) and Hla. In summary, our findings establish the strict inducibility and functional relevance of the NLRP3 inflammasome in non-myeloid, epithelial cells in vitro. These results resolve conflicting reports and position keratinocytes as a context-specific, non-hematopoietic cellular model for studying NLRP3 activation in host-microbe interactions at barrier tissues. KEY FINDINGS Additional evidence that NLRP3 is absent in resting, nonstimulated human keratinocytes in vitro IFNγ, but not LPS, is a potent ‘priming’ signal for NLRP3 in human keratinocytes in vitro In IFNγ-primed keratinocytes, S. aureus α-hemolysin (Hla) selectively activates NLRP3, whereas nigericin activates both NLRP1 and NLRP3 in vitro SAg and Hla kill keratinocytes via NLRP3-driven pyroptosis in the presence of T cells in vitro GRAPHICAL ABSTRACT

Summary Aneuploidy—defined as gains and losses of chromosomes—is frequently observed in cancer and has been implicated in promoting tumor progression and metastasis. However, the molecular mechanisms underlying this phenomenon remain poorly understood. By generating new in vitro and in vivo models of aneuploidy, we found that aneuploidy confers remarkable resistance to reactive oxygen species (ROS)-mediated cell death. This is a general consequence of aneuploidy, independent of the specific chromosomes gained or lost. Mechanistically, aneuploidy-induced resistance to cell death results from suppressed Poly(ADP-Ribose) Polymerase 1 (PARP1) in aneuploid cells, which inhibits PARP1-mediated cell death after ROS (parthanatos). We validated aneuploidy-associated PARP1 suppression across 15 cell models and human tumors, with pronounced effects in metastatic tumors. Importantly, decreased PARP1 levels in aneuploid cells promote tumor metastasis and vice versa. Through genome-wide CRISPR screen, a focused CRISPRa screen and functional validation, we identified the transcription factor CCAAT/enhancer-binding protein beta (CEBPB) as a critical mediator of PARP1 downregulation and ROS resistance in aneuploid cells. Furthermore, we found lysosomal dysfunction as the upstream mediator of CEBPB activation in aneuploid cells. We propose that during tumorigenesis, aneuploidy-driven CEBPB activation promotes PARP1 suppression fostering ROS resistance and cancer progression. Highlights Aneuploidy universally confers resistance to oxidative stress independent of p53 status, karyotype and cell lineage through inhibition of PARP1 expression and activity Suppressed PARP1 enhances metastatic potential, while PARP1 restoration suppresses metastatic spread, revealing a novel mechanism linking aneuploidy to cancer progression. PARP1 suppression compromises DNA damage repair and cell death to multiple genotoxic stressors, including reactive oxygen species, alkylating agents, and UV radiation. A genome-wide CRISPR screen and a CRISPRa screen identifies CEBPB as the critical transcription factor mediating PARP1 regulation and ROS resistance. Nuclear CEBPB increases significantly after aneuplodization in experimental systems and in scRNAseq of primary human cancer patients.

Recursive splice sites are rare motifs postulated to facilitate splicing across massive introns and shape isoform diversity, especially for long, brain-expressed genes. The necessity of this unique mechanism remains unsubstantiated, as does the role of recursive splicing (RS) in human disease. From analyses of rare copy number variants (CNVs) from almost one million individuals, we previously identified large, heterozygous deletions eliminating an RS site (RS1) in the first intron of CADM2 that conferred substantial risk for attention deficit hyperactivity disorder (ADHD) and other neurobehavioral traits. CADM2 encodes a neuronally expressed cell adhesion molecule that has repeatedly been associated with ADHD and numerous similar traits. To explore the molecular impact of RS ablation in CADM2 , we used CRISPR to model patient deletions and to target a smaller region (~500 base pairs) containing RS1 in both human induced neurons (iNs) and rats. Transcriptome analyses in unedited iNs provided a catalog of CADM2 transcripts, including novel transcripts that retained RS exons. Intriguingly, ablating RS1 altered the gradient of RNA abundance across the first intron of CADM2 , decreased the level of CADM2 expression, and impacted transcript usage. Decreased CADM2 expression was reflected in reduced exon usage downstream of the RS1 site and global alteration to genes involved in neuronal processes including synapse and axon development. Given the scale of our analyses and the widespread association of CADM2 with neurobehavioral traits, we sought to validate these findings using in vivo models and found that rodent models harboring Cadm2 RS1 deletions exhibited significant changes in relevant behaviors and functional brain connectivity. In summary, our analyses demonstrate a functional role for RS as a noncoding regulatory mechanism in a gene associated with a spectrum of neuropsychiatric and behavioral traits.

Genome-wide association studies (GWAS) have contributed significantly to unraveling the genetic bases of complex diseases such as Parkinson’s disease (PD); yet experimental evidence for causation is often elusive. Here, we hypothesized that non-manifesting carriers of a PD-causing mutation in the LRRK2 gene could express genetic modifiers conferring disease protection. Using a pluripotent stem cell-based model, we showed that dopaminergic neurons derived from these individuals were partially protected from the disease in vitro, and that this protective effect is genetically driven. Whole-exome sequencing identified a previously unreported low-frequency variant in cyclin G-associated kinase (GAK) that was associated with a nearly nine-year delay in age at onset among LRRK2 mutation carriers in a local cohort, although replication in additional cohorts was inconclusive. To rule out inter-cohort heterogeneity, we used CRISPR/Cas9-mediated gene editing to isolate the effect of the mutation. We found that the candidate protective variant prevented neuron loss in vitro along with an improvement of several indicators endocytic-mediated transport. Together, our findings provide mechanistic insights into PD pathogenesis and actionable genetic information for the prognosis of PD patients. One Sentence Summary Investigating genetic protection against Parkinson’s disease in non-manifesting carriers of LRRK2 mutations by CRISPR/Cas9-based genome edition.

ABSTRACT Advances in single-cell sequencing have deepened our understanding of cellular identities. However, because they inherently capture only static snapshots, after which no further observations are possible, we cannot compare past and present profiles within the same cell. Thus, multi-time-point whole-genome profiling at single-cell resolution has been a long-standing goal. Here, we introduce the History Tracing-sequencing (HisTrac-seq) platform, which enzymatically labels genomic DNA adenine to “bookmark” gene regulatory statuses. This first enabled the profiling of transcriptomic and epigenetic states in the mouse brain over a period of two months. Furthermore, extending HisTrac-seq to single-cell multi-omics sequencing, we demonstrated the simultaneous mapping of past and present profiles of the same single cells. Analyzing over 93,000 cells, we discovered unexpected, drastic cell identity transitions on a large scale (“identity jumps”). This phenomenon was previously unobservable with current technologies and revealed a hidden layer of developmental plasticity. HisTrac-seq offers a powerful approach to “temporal multi-omics” for disentangling dynamic biological processes involved in development, plasticity, aging, and disease progression.

ABSTRACT The cytokines interleukin (IL)-22 and IL-17 are secreted by innate and adaptive immune cells to drive “type III” responses that protect against extracellular pathogens, promote mucosal barrier integrity, and foster microbiota homeostasis. However, dysregulation of IL-22 and/or IL-17 contributes to autoimmunity, chronic inflammation, and malignancy. Thus, a deeper understanding of mechanisms regulating type III cytokine production could provide new therapeutic targets for a spectrum of immune-mediated diseases. Toward this goal, we performed a genome-wide CRISPR inhibition (CRISPRi) screen to identify factors that regulate IL-22/IL-17 expression in a murine type III innate lymphoid cell (ILC3) model, MNK3, following stimulation with IL-23 and IL-1b. In addition to previously known regulators of type III cytokines, including IL-23 receptor components IL23R and IL12RB1, the screen identified a large set of new factors that either potentiate or attenuate expression of IL-22 and/or IL-17. A subset of these novel factors was chosen for validation, from which two were selected for further study. The nuclear protein, SON, which binds both DNA and RNA, impaired expression of IL12RB1 at the levels of de novo transcription and RNA processing. The second, MAP4K1 (HPK1), is a serine/threonine kinase that is required for IL-22 but not IL-17 expression. Depletion of MAP4K1 in MNK3 also enhanced expression of the type I cytokine, IFNg, which was co-expressed with IL-17, a phenotype reminiscent of pathogenic Th17 cells. Together, results from the CRISPRi screen broaden our understanding of the factors involved in type III immune responses and offer new targets for modulating IL-22/17 expression.

CRISPR-based genetic perturbation screens have revolutionized the ability to link genes to cellular phenotypes with unprecedented precision and scale. However, conventional pooled CRISPR screens require large cell numbers to achieve adequate sgRNA representation, posing technical and financial challenges. Here, we investigate the impact of co-delivery of multiple guide RNAs via high multiplicity of infection (MOI) in pooled CRISPR interference (CRISPRi) screens as a strategy to enhance screening efficiency while reducing cell numbers. We systematically evaluate screen performance across varying MOIs, assessing the effects of multiplexing on knockdown efficiency, sgRNA representation, and potential interference of multiple sgRNA phenotypes. Our data demonstrate that sgRNA multiplexing (MOI 2.5-10) can maintain screen performance while enabling significant reductions in cell number requirements. We further apply these optimized conditions to conduct a genome-wide CRISPR screen for regulators of the intracellular adhesion molecule ICAM-1, successfully identifying novel candidates using as few as half a million cells. This study provides a framework for adopting multiplexed sgRNA strategies to streamline CRISPR screening applications in resource-limited settings.

Anopheles stephensi is a major malaria vector mainly present in southern Asia and the Arabian Peninsula. Since 2012 it has invaded several countries of eastern Africa, stimulating urgent efforts to develop more efficient strategies for vector control such as CRISPR/Cas9-based homing gene drives. Target site resistance is a significant challenge to the deployment of these systems. When a double-stranded break is repaired by NHEJ, it can lead to mutations which destroy the target site, making that allele unrecognizable to the sgRNA and resistant to further cleavage. The use of multiple sgRNAs has the potential to solve this issue. We performed experimental crosses to assess the homing and cutting efficiency of two different multiplexing strategies targeting the cardinal locus, in the presence and absence of a resistance allele. We found pre-existing mutations at one sgRNA target site did not significantly reduce the homing efficiency for either strategy. Modelling indicates that while both strategies can overcome resistance allele formation, the fitness of the drive-carrying alleles is a critical factor in determining the overall performance and persistence of a split drive.

Abstract Rifampin is the most effective drug in the treatment of tuberculosis.However, certain strains of MTB have developed resistance to rifampin, leading to the need for alternative treatment options.The rpoB gene mutations play a central role in MTB resistance to the rifampin, so it is essential to identify these mutations and efficiently treat rifampin-resistant MTB strains.This study developed a novel CRISPR-Cas12a platform integrated with recombinase polymerase amplification (RPA) and fluorescence detection to specifically identify the rpoB _L378R mutation associated with Rifampin resistance in (MTB).We found that this detection system was highly specific and did not cross-react with created reference samples containing the genomes of MTB H37Rv, Mycobacterium smegmatis, Mycobacterium aureus , and Escherichia coli. The CRISPR-Cas12a-based platform developed in this study was simple, sensitive, and specific for detecting the Rifampin-resistant MTB strain with the rpoB _L378R mutation.This suggested that it has potential for clinical applications in identifying MTB rpoB _L378R mutation.

Protein homeostasis is tightly controlled by the coordinated actions of E3 ubiquitin ligases and deubiquitinases (DUBs). We identify Spindlin-4 (SPIN4), a histone H3K4me3 reader, as a substrate regulated by opposing pathways: degradation mediated by the Cullin-RING E3 ligase DCAF16 and stabilization by the DUB BAP1. Through CRISPR-Cas9 knockout screens and biochemical analyses, we demonstrate that DCAF16 promotes SPIN4 degradation, while BAP1 interacts with and stabilizes SPIN4 through its catalytic activity. Inhibition or loss of BAP1 reduces SPIN4 levels, highlighting its critical role in maintaining SPIN4 homeostasis. Proteomics and interactome analyses further support this regulatory axis. These findings reveal a dynamic balance controlling SPIN4 stability, with potential implications for epigenetic regulation and disease processes.

ABSTRACT Functional genomics screens have illuminated genetic dependencies in cancer, but conventional in vitro approaches fail to capture vulnerabilities shaped by the tumor microenvironment. Here, we implement CRISPR-StAR (Stochastic Activation by Recombination), a next-generation inducible CRISPR screening platform for large-scale in vivo applications. The system uses a dual lox-based recombination system to enable guide-level normalization and clonal knockout phenotyping. To analyze the rich (barcode-embedded) sequencing output, we developed UMIBB, a superior Bayesian statistical framework for quantifying gene-level dropout and enrichment compared to conventional software packages. Screening a 30,000-sgRNA library in A549 xenografts, followed by clone representation and dropout correlation analyses, showed high fidelity and reproducibility with dropout phenotypes resolvable using as few as 30 tumors for this size library. Validation across multiple tumor models demonstrated that a single tumor can provide reliable, functional annotation for ∼1,000 genes leveraging intra-tumor library controls for normalization. Comparing in vivo and in vitro screens revealed that a substantial subset of tumor suppressor genes exerts strong phenotypic effects only observable in vivo . For example, single-gene knockout and transcriptomic profiling confirmed that KMT2C and KMT2D have contrasting impacts on tumor growth - an insight that would have been overlooked in standard cell culture. Looking ahead, CRISPR-StAR screening, combined with our user-friendly analysis pipeline available on GitHub (R-package), offer an integrated framework for creating in vivo dependency maps that can complement existing vitro datasets like DepMap and Achilles. Critically, our approach reduces animal use by up to 7-fold compared to conventional in vivo dropout screens. This represents a significant ethical and methodological advancement - achieving genome-scale resolution with far fewer animals and greater reproducibility.

Miniature CRISPR-Cas12f nucleases are attractive candidates for therapeutic genome editing owing to their compact size and compatibility with adeno-associated virus (AAV) delivery. However, editing efficiencies in mammalian cells are lower than those of larger systems such as Cas12a and SpCas9. The extensive phylogenetic diversity of Cas12f suggests unexplored mechanistic variation with the potential for optimization. Here, we characterize a naturally occurring Cas12f ortholog discovered through metagenomics, Cas12f-MG119-28, which supports robust genome editing in human cells. Through structural, biochemical, and kinetic analyses, we compare Cas12f-MG119-28 with two recently described orthologs, Oscillibacter sp. Cas12f (OsCas12f) and Ruminiclostridium herbifermentans Cas12f (RhCas12f). These orthologs present divergent architectures and regulatory features governing PAM recognition, gRNA binding, dimerization, and DNA cleavage. Notably, Cas12f-MG119-28 achieves efficient R-loop formation via a stable dimer interface and a naturally optimized guide RNA. These discoveries elucidate key mechanistic determinants of Cas12f activity and may offer a framework for engineering compact genome editors with therapeutic potential.

Identifying new genes responsible for non-syndromic hearing loss remains a critical goal, as many individuals with hereditary deafness still lack a molecular diagnosis despite comprehensive genetic testing. The tectorial membrane (TM) is a specialized, collagen-rich, acellular matrix of the inner ear, essential for stimulating mechanosensitive hair cell bundles during sound transduction, and its structural integrity is critical for frequency tuning and auditory sensitivity. Although mutations in genes encoding a number of non-collagenous proteins found in the TM (TECTA, CEACAM16, OTOG, OTOGL) have been identified as deafness genes, definitive evidence implicating β-tectorin (TECTB) in human hearing loss has been lacking. Here, we present multiple lines of genetic and experimental evidence linking a missense variant in TECTB (c.674G>A, p.Cys225Tyr) to autosomal dominant, non-syndromic hearing loss in a multigenerational family. The variant alters one of eight highly conserved cysteines present within the zona pellucida (ZP) domain of TECTB and is predicted to disrupt protein folding and matrix assembly. Using a Tectb-C225Y knock-in mouse model, we show that homozygous animals exhibit severe hearing loss and profound disruption of TM morphology, while heterozygote animals display decreased matrix content within the TM and increased susceptibility to noise-induced hearing loss—despite normal auditory thresholds. These findings identify TECTB as a novel human deafness gene, further elucidate its structural role in maintaining TM integrity, and highlight its contribution to resilience against environmental and age-related auditory decline.

Summary A pause in DNA synthesis that occurs when the replisome encounters an obstacle could lead to genome instability. Although important, systematic identification of replication pause sites is challenging due to their low frequency and delocalized nature. Here we present the first single-molecule identification of sites of replisome perturbation across a eukaryotic genome using long-read nanopore sequencing. For each single-molecule replication pause we determine the direction of replication, leading/lagging-strand identity, location and approximate duration, and whether the replisome resumed synthesis. Although pauses are largely diffuse over the genome, they are significantly enriched over transcribed features and correlate with transcription and R-loop levels. Transcription-replication conflicts are more numerous when head-on than co-directional. Finally, we identified genomic loci with a strong bias towards leading over lagging strand pauses, consistent with uncoupling of the helicase from polymerase epsilon. Our data support helicase-polymerase uncoupling resulting from replication pausing as the molecular trigger behind epigenetic switching. Graphical abstract

Background The homozygous ADA2 c.506G>A (p.Arg169Gln; p.R169Q) variant accounts for majority of Deficiency in Adenosine Deaminase 2 (DADA2). This monogenic disorder may be amenable to ex vivo gene therapy by correcting the pathogenic mutation in CD34+ hematopoietic stem and progenitor cells (HSPCs). Objective To apply CRISPR-Cas9 and homology-directed repair (HDR) as a surrogate strategy to model correction of the pathogenic ADA2 c.506G>A variant in healthy cord blood HSPCs. Methods HSPCs were electroporated with optimised CRISPR-Cas9 editing reagents, and editing outcomes, including HDR and on-target deletions, were quantified by ddPCR. Cell functionality was assessed through colony-forming unit (CFU) assays and by xenotransplantation into NOD SCID Gamma (NSG) mice. Two HDR enhancement strategies were tested: (1) genetic inhibitors of p53 and non-homologous end joining (NHEJ) pathways, and (2) pharmacological NHEJ inhibition. Results Small-molecule NHEJ inhibitors increased HDR efficiency approximately two-fold (from ∼40 % to ∼80 %). Edited HSPCs retained normal CFU capacity and successfully engrafted in NSG mice. However, up to 8 % of edited cells exhibited on-target chromosome loss, though this declined over time. Up to 40 % of T cells and fibroblasts demonstrated similar losses under NHEJ inhibitors treatment. In contrast, genetically encoded inhibitors did not improve HDR. Conclusion The ADA2 p. c.506G>A variant can be effectively edited employing surrogate strategy in HSPCs without impairing functionality. Although pharmacological inhibition of NHEJ enhances HDR efficiency, it also increases the risk of on-target chromosome aberrations, highlighting the need for careful consideration of the associated risks and benefits in therapeutic gene editing. Key messages 1) The ADA2 p.R169Q variant can be efficiently corrected via HDR, and the edited CD34+ HSPCs retain their engraftment capability in NSG mice. 2) Pharmacological inhibition of NHEJ using small-molecule inhibitors increases HDR efficiency but is associated with significant on-target deletions and chromosomal arm loss, particularly in differentiated cell types, and in a donor-dependent manner. Capsule summary The ADA2 p.R169Q variant is a viable target for precision gene editing in hematopoietic stem cells. Although inhibition of NHEJ improves HDR efficiency, it concomitantly increases the risk of large on-target deletions, particularly in differentiated cells.

Fusarium wilt, caused by Fusarium oxysporum f. sp. vasinfectum (Fov), is one of the most destructive early-season cotton diseases worldwide. The recent emergence of the highly virulent Fov race 4 (Fov4) and its aggressiveness have raised significant concerns for the U.S. cotton industry. Unlike predominant Fov races in US cotton production, which require root-knot nematodes to cause damage, Fov4 is known to infect cotton independent of nematodes. However, molecular mechanisms of Fov4 virulence in cotton are not clearly understood. Secondary metabolites are often identified as the culprits in pathogen virulence toward plant hosts. To investigate these factors in Fov4, we analyzed the genomes of Fov1 and Fov4 using Fungal antiSMASH and identified a Fov4-specific nonribosomal peptide synthetase (NRPS) gene FNP1 . To investigate its function, we generated FNP1 knock-out mutant using CRISPR-Cas9 approach. Growth assays revealed that the mutants exhibit significantly attenuated hyphal production on media containing cotton roots as the sole carbon source, increased sensitivity to cell stress agents, as well as lagged spore germination. Furthermore, the mutant exhibited defect in cotton root rot virulence and significant decrease in Fusaric acid production. Microscopic observation of GFP-labeled FNP1 deletion mutant showed impeded infection progression in cotton roots compared to the wild type (WT), which further explained the impeded virulence in FNP1 mutant. Gene complementation restored the observed defects, confirming that FNP1 is critical for Fov4 virulence, hyphal development, Fusaric acid production, and stress responses. Highlights Comparative genomic analysis between Fov1 and Fov4 identified FNP1 as a gene specific to Fov4. CRISPR/Cas9 system was employed in Fov4 to generate gene deletion mutants and GFP labeling. FNP1 plays a critical role in Fov4 hyphal development, virulence, fusaric acid production, and stress responses. This study is the first report to identify and functionally characterize a virulence gene in Fusarium oxysporum f. sp. vasinfectum (Fov) race 4 (Fov4) in cotton wilt pathogenesis.

SUMMARY Cas9 nucleases are the effectors of the class 2 type II CRISPR system in bacteria and function to restrict invading DNA. They can also be used with single guide RNAs (sgRNAs) as antimicrobials and genome engineering tools in bacteria, yet applications are hindered by an incomplete understanding of Cas9-target interactions. Here, we generate large-scale SaCas9/sgRNA in vivo bacterial activity datasets and train a machine learning model (crisprHAL) to predict SaCas9 activity. The highest predictive performance was found when downstream sequence flanking the canonical NNGRRN PAM motif at positions [+1] and [+2] was included in model training, correlating with high in vivo activity on sites that included T-rich di-nucleotides in the [+1] and [+2] flanking positions. Strikingly, model predictions and experimentally determined activity in pooled sgRNA experiments in Escherichia coli and Citrobacter rodentium showed an ∼10-fold reduced SaCas9 activity at sites with 5′-NNGGAT[C]-3 ′ PAM [+1] sequences. Cleavage assays using plasmid DNA isolated from E. coli inactivated for DNA adenine methyltransferase (DAM) and SaCas9/sgRNA combinations targeting sites with NNGGAT[C] PAM sequences confirmed that adenine methylation impacts SaCas9 cleavage. Moreover, ablation of a GATC DAM site in a PAM sequence enhanced SaCas9 in vitro activity, whereas creation of a DAM site reduced activity, providing a mechanistic link between adenine methylation and SaCas9 activity. Our results show that a general purpose machine learning architecture can provide biologically relevant insights into SaCas9-PAM interactions that can better inform activity predictions for bacterial applications. Avoidance of adenine methylated PAM sites by SaCas9 may be a mechanism of self versus non-self discrimination or reflect an evolutionary adaptation to counter methylation as an anti-restriction strategy by phage or plasmids.

Conditional sex transformation systems are promising tools in the fight against insect pests. In this study, we developed and tested CRISPR-based, tetracycline-repressible sex transformation strains in the Australian sheep blowfly, Lucilia cuprina . Two CRISPR effector molecules, Cas9 and dCas9, were employed to target the sex-determining gene transformer with the goal of turning female blowflies into males. The Cas9 version of the system induced robust knockout of a visual marker gene but failed to trigger sex transformation without external provision of transformer -targeting sgRNAs. Furthermore, we found that dCas9 expression was linked to several deleterious phenotypes, including developmental delays, reduced body weight, and death. Our study provides the first proof-of-concept conditional CRISPR systems in L. cuprina , and suggests that while dCas9 is toxic at high levels in this species, Cas9 is well-tolerated and may be able to induce sex transformation with minor modifications to the system.

The efficiency and specificity of guide RNAs continue to be crucial obstacles for successful experimental design, despite the fact that CRISPR/Cas9 has transformed genome editing. In this work, we introduce a computational method for optimizing CRISPR/Cas9 guide RNA that combines PAM diversity, local efficiency penalties, and weighted off-target scoring to find high-performing guides across a range of genome compositions. To capture a variety of natural genomic complexity, we simulated five sample genomes: AT-rich, GC-rich, balanced GC content, and high-repeat variations. All twenty-nucleotide target sequences were scanned for each genome, and off-target potential was assessed by permitting up to two mismatches with weighted penalties for seed region sites. To accommodate for any secondary structure impacts, efficiency assessment included both local sliding window penalties and global GC content. Furthermore, we looked at several PAM sequences that were pertinent to various Cas9 variations in order to assess how they affected guide selection. The findings show that efficiency scores vary by genome composition, with the highest scoring guides consistently displaying zero anticipated off-target events. While balanced genomes showed intermediate tendencies, GC-rich genomes tended to choose slightly higher efficiency guides than AT-rich genomes. PAM type affects guide efficiency, according to analysis across several genomes, and the combination of efficiency and off-target score consistently indicates guides with good expected performance. Three-dimensional scatter plots of efficiency and off-target counts versus genomic position, violin plots of off-target distributions, and genome-wide heatmaps emphasizing the best guide positions were used to illustrate these findings. In addition to offering a generalizable computational method for choosing CRISPR/Cas9 guides that optimize specificity and efficiency, our study gives fresh insights into the interactions among genome composition, PAM selection, and guide design criteria. By taking into account weighted off-target penalties, genome complexity, and local efficiency effects, this in silico framework overcomes some of the main drawbacks of earlier simulations. It is also easily applicable to direct selection for experimental research on a variety of organisms. The results provide the groundwork for future advancements in genome editing techniques by establishing a predictive computational framework that can expedite CRISPR/Cas9 research and minimize trial and error in guide selection.

Abstract CDKL5 deficiency disorder (CDD) is a severe X-linked neurodevelopmental condition characterized by early-onset epilepsy, intellectual disability, and motor dysfunction. Here, we present a dual-effector CRISPR-based epigenome editing platform that enables targeted reactivation of the silenced CDKL5 allele. Using a split dCas9 system compatible with AAV9 delivery, we achieved simultaneous transcriptional activation and DNA demethylation of the CDKL5 promoter in both murine and human models of disease. In heterozygous Cdkl5 E6del mice, intracerebroventricular delivery of the editor restored Cdkl5 protein expression, re-engaged downstream signaling, and rescued motor and cognitive deficits. In patient-derived neural stem cells and cortical organoids, CDKL5 reactivation normalized gene expression and restored neuronal network activity. These findings establish a scalable, mutation-independent strategy for treating CDD and highlight the therapeutic potential of epigenetic reprogramming for X-linked disorders.