ABSTRACT Double-strand breaks (DSBs) are toxic lesions that lead to genome instability. While canonical DSB repair pathways typically operate independently of RNA, emerging evidence suggests that RNA:DNA hybrids and transcripts near damaged sites can influence repair outcomes. However, a direct role for transcript RNA as a template during DSB repair in human cells is yet to be established. In this study, we designed fluorescent- and sequencing-based assays, which demonstrated that RNA-containing oligonucleotides and messenger RNA serve as templates to promote DSB repair. We conducted a CRISPR/Cas9-based genetic screen to identify factors that promote RNA-templated DSB repair (RT-DSBR), and of the candidate polymerases, we identified DNA polymerase-zeta (Polζ) as the potential reverse transcriptase that facilitates RT-DSBR. Furthermore, by analyzing sequencing data from cancer genomes, we identified the presence of whole intron deletions, a unique genomic scar reflective of RT-DSBR activity generated when spliced mRNA serves as the repair template. These findings highlight RT-DSBR as an alternative pathway for repairing DSBs in transcribed genes, with potential mutagenic consequences.

Background: Gene editing techniques offer new opportunities to improve important traits in aquaculture. The allergenicity of fish flesh is a major problem in aquaculture. Parvalbumin (Parv) is the most prevalent fish allergen. For instance, in salmonids, a single parvalbumin beta-1 protein (parvb1) has been identified as an allergen in specific patients. Therefore, generating trout carrying two parvb1 alleles deleted from the allergenic peptide-encoding region could prevent allergies in these sensitive individuals. Methods Here, we describe the application of the Crispr/cas9 system in an attempt to delete parvb1 exon 2 encoding the allergenic peptide and, alternatively, to replace exon 2 of parvb1 with exon2 of parvalbumin beta-2 protein (parvb2,) which does not encode the allergenic peptide. Exon skipping and swapping were pursued through microhomology-mediated end-joining (MMEJ) knock-In using specifically designed double-stranded donor DNA. Results Genotyping of approximately 200 F0 fingerlings originating from eggs injected with donor DNA designed for exon 2 skipping led to the identification of only one animal carrying an allele lacking exon 2. Genotyping of approximately 150 fingerlings originating from eggs injected with donor DNA for exon 2 swapping did not result in any trout carrying the expected modified allele. Conclusions These preliminary results indicate the potential difficulties associated with the MMEJ KI experiments performed in farmed fish. Finally, new genomic techniques in aquaculture are further discussed in the context of lively debates taking place in the European parliament regarding a possible revision of the current law that determines the legal status of farm animals modified by genome editing. Gene editing, microhomology-mediated end-joining knock-in, parvalbumin, allergenicity, trout, and genetically modified organisms (GMOs).

Loop-extrusion machinery, comprising the cohesin complex and CCCTC-binding factor CTCF, organizes the interphase chromosomes into topologically associating domains (TADs) and loops, but acute depletion of components of this machinery results in variable transcriptional changes in different cell types, highlighting the complex relationship between chromatin organization and gene regulation. Here, we systematically investigated the role of 3D genome architecture in gene regulation in mouse embryonic stem cells under various perturbation conditions. We found that acute depletion of cohesin or CTCF disrupts the formation of TADs, but affects gene regulation in a gene-specific and context-dependent manner. Furthermore, the loop extrusion machinery was dispensable for transcription from most genes in steady state, consistent with prior results, but became critical for a large number of genes during transition of cellular states. Through a genome-wide CRISPR screen, we uncovered multiple factors that can modulate the role of loop extrusion machinery in gene regulation in a gene-specific manner. Among them were the MORF acetyltransferase complex members (Kat6b, Ing5, Brpf1), which could antagonize the transcriptional insulation mediated by CTCF and cohesin complex at developmental genes. Interestingly, inhibition of Kat6b partially rescues the insulator defects in cells lacking the cohesin loader Nipbl, mutations of which are responsible for the developmental disorder Cornelia de Lange syndrome. Taken together, our findings uncovered interplays between the loop extrusion machinery and histone modifying complex that underscore the context-dependent and gene-specific role of the 3D genome.

Summary Genome editing using CRISPR/Cas is a key technology for speeding up breeding for climate-resilient, high-yielding crops (Scheben et al ., 2017). However, efficient targeted mutagenesis requires implementing stable transformation methods and establishing a CRISPR/Cas setup suitable for the species of interest (Shan et al ., 2020). The availability of such methods is a significant bottleneck to advancing many promising, albeit under-researched, crops. Testing and establishing vectors for efficient application of CRISPR/Cas in non-model crops could boost research and breeding of new valuable crops (Ye and Fan, 2021). We edited key pathway genes in the betalain biosynthesis pathway of grain amaranth, i.e., A. hypochondriacus L ., to prove how targeted mutagenesis can be implemented in an orphan crop using the CasCADE modular cloning system (Hoffie, 2022). Grain amaranth is a resilient C 4 dicot orphan crop with excellent nutritional composition. These properties make amaranth a well-suited candidate to be bred as a climate-resilient crop (Joshi et al ., 2018). However, no efficient and reproducible protocol for successful application of CRISPR/Cas9 or stable transformation and regeneration, has been demonstrated in A. hypochondriacus (Castellanos-Arévalo et al ., 2020).

ABSTRACT The axonal membrane-associated periodic skeleton (MPS), consisting of F-actin rings crosslinked by spectrin heterotetramers, is ubiquitous and critical for neuronal function and homoeostasis. However, the initiation and early development of the axonal MPS are poorly understood. Using superresolution imaging, we show that βII-spectrin is recruited early to the axonal cortex, followed by progressive establishment of long-range periodic order. Microtubule dynamics are essential for MPS formation in the early stages, but transition to a passive stabilising role in mature axons. We show that the early subplasmalemmal recruitment of βII-spectrin is dependent on cortical actin but not on actomyosin contractility, and active nucleation of F-actin is required in early development but is dispensable for the mature MPS. Using a βII-spectrin knockout model, we demonstrate that the actin-binding and lipid-interacting domains of βII-spectrin are critical for its subplasmalemmal confinement and, subsequently, MPS maturation. These findings highlight stage-specific cytoskeletal remodelling underlying MPS development and advance our understanding of axonal subcellular architecture.

Wound infection is a major disruptor of wound healing. Keratinocytes, critical in repair and microbial responses, require the L-arginine hydrolysing enzyme arginase1, for effective healing. Wound pathogens such as Pseudomonas aeruginosa may also need L-arginine. We therefore investigated host-microbial interactions in the context of wound healing and L-arginine metabolism. Arginase-inhibited murine wounds challenged with P. aeruginosa exhibited significantly delayed re-epithelialisation. This finding was recapitulated in vitro using P. aeruginosa- challenged, arginase1 deficient ( shARG1) keratinocytes associated with reduced epithelial proliferation and viability, and heightened inflammation. Whilst P. aeruginosa challenge promoted host metabolism of L-arginine, this was perturbed in wounded shARG1 keratinocytes. There was, however, heightened downstream polyamine metabolism in shARG1 cells when under P. aeruginosa challenge. Host keratinocyte arginase1 deficiency promoted bacterial growth in vitro , in line with a failure to upregulate the anti-microbial peptides, β-defensins, in shARG1 scratches. This work demonstrates a pivotal role for keratinocyte arginase1 in wound infection.

Interferons (IFN) are cytokines that regulate the expression of hundreds of genes during viral infections to generate a broadly antiviral environment in the stimulated cell. Antiviral breadth is provided by the concurrent expression of many individual IFN-stimulated genes (ISG), each encoding a protein with often exquisite antiviral specificity. Here, we show that mechanistic plasticity at a single genetic locus is a novel mechanism to diversify the antiviral profile of human cells. Through alternative splicing, the OAS2 gene encodes two antiviral molecules with distinct target specificities. The shorter OAS2 p69 isoform blocks the replication of seasonal human coronavirus OC43 (HCoV-OC43), while the longer p71 isoform restricts the replication of picornavirus Cardiovirus A (EMCV). The restriction profile is determined by the variable length OAS2 C-terminal tail. Remarkably, the antiviral mechanisms underlying these distinct antiviral profiles are either RNase L dependent or independent, suggesting that splicing divides ‘classic restriction’ versus ‘virus sensing’ systems across two distinct OAS2 polypeptides. Together, our data reveal that the human OAS2 locus uses alternative splicing and mechanistic plasticity to diversify antiviral profiles.

ABSTRACT To gain insight into biological mechanisms that cause resistance to DNA damage, we performed parallel pooled genetic CRISPR-Cas9 screening for survival in high risk HNSCC subtypes. Surprisingly, and in addition to ATM, DNAPK, and NFKB signaling, JAK1 was identified as a driver of tumor cell radiosensitivity. Knockout of JAK1 in HNSCC increases cell survival by enhancing the DNA damage-induced G2 arrest, and both knockout and JAK1 inhibition with abrocitinib prevent subsequent formation of radiation-induced micronuclei. Loss of JAK1 function does not affect canonical CDK1 signaling but does reduce activation of PLK1 and AURKA, kinases that regulate both G2 and M phase progression. Correspondingly, JAK1 KO was found to cause mitotic defects using both EdU labeling and live cell imaging techniques. Given this insight, we evaluated Kif18a inhibition as an approach to exacerbate mitotic stress and enhance the efficacy of radiation. These studies establish Kif18a inhibition as a novel strategy to counteract therapeutic resistance to DNA damage mediated by G2 cell cycle arrest.

Design: ing drugs that can restore a diseased cell to its healthy state is an emerging approach in systems pharmacology to address medical needs that conventional target-based drug discovery paradigms have failed to meet. Single-cell transcriptomics can comprehensively map the differences between diseased and healthy cellular states, making it a valuable technique for systems pharmacology. However, single-cell omics data is noisy, heterogeneous, scarce, and high-dimensional. As a result, no machine learning methods currently exist to use single-cell omics data to design new drug molecules. We have developed a new deep generative framework named MolGene-E to tackle this challenge. MolGene-E combines two novel models: 1) a cross-modal model that can harmonize and denoise chemical-perturbed bulk and single-cell transcriptomics data, and 2) a contrastive learning-based generative model that can generate new molecules based on the transcriptomics data. MolGene-E consistently outperforms baseline methods in generating high-quality, hit-like molecules from gene expression profiles obtained from single-cell datasets as validated by target knock-out experiments using CRISPR. This superior performance is demonstrated across diverse de novo molecule generation metrics. Extensive evaluations demonstrate that MolGene-E achieves state-of-the-art performance for zero-shot molecular generations. This makes MolGene-E a potentially powerful new tool for drug discovery.

Precise control over the dosage of Cas9-based technologies is essential because off-target effects, mosaicism, chromosomal aberrations, immunogenicity, and genotoxicity can arise with prolonged Cas9 activity. Type II anti-CRISPR proteins (Acrs) inhibit and control Cas9 but are generally impermeable to the cell membrane due to their size (6–54 kDa) and anionic charge. Moreover, existing Acr delivery methods are long-lived and operate within hours ( e.g ., viral and non-viral vectors) or are not applicable in vivo ( e.g ., nucleofection), limiting therapeutic applications. To address these problems, we developed the first protein-based anti-CRISPR delivery platform, LF N -Acr/PA, which delivers Acrs into cells within minutes. LF N -Acr/PA is a nontoxic, two-component protein system derived from anthrax toxin, where protective antigen proteins bind receptors widespread in human cells, forming a pH-triggered endosomal pore that LF N -Acr binds and uses to enter the cell. In the presence of PA, LF N -Acr enters human cells at concentrations as low as 2.5 pM to inhibit up to 95% of Cas9-mediated knockout, knock-in, transcriptional activation, and base editing. Timing LF N -Acr delivery reduces off-target base editing and increases Cas9 specificity by 41%. LF N -Acr/PA is the most potent known cell-permeable CRISPR-Cas inhibition system, significantly improving the utility of CRISPR for genome editing.

Fluorescence microscopy has become an indispensable tool in biological research, offering powerful approaches to study protein dynamics and molecular biochemistry in vivo . Among archaea, Haloferax volcanii has emerged as a particularly well-suited model organism for imaging studies, with a growing toolkit of established fluorescent markers, plasmids, and promoter systems. Recent advances in single-molecule imaging techniques have created new opportunities through WR806, a carotenoid-free strain providing reduced autofluorescence background. However, existing plasmid-based expression systems in WR806 show critical limitations in protein expression control and challenges with protein aggregation. To address these limitations, we developed pUE001, a novel expression system specifically designed for WR806. This system achieves precise expression control by decoupling selection and induction through strategic implementation of the trpA selection marker. Through comprehensive characterization, we demonstrate that pUE001 provides superior control over protein expression compared to the previously established pTA962 system. It enables linear, titratable expression of diverse proteins - from the highly regulated CRISPR-Cas component Cas1 to the abundant structural protein FtsZ1 - while preventing protein aggregation that could compromise native cellular functions. Additionally, we performed a comprehensive analysis of WR806 to show that carotenoid depletion does not affect native cellular physiology. Finally, to demonstrate the system's utility, we investigated the role of Cas1 in UV-induced DNA repair using single-particle tracking photoactivated localization microscopy (sptPALM). Our findings reveal significant, dose-dependent changes in Cas1 mobility following UV-light induced damage, providing evidence for its involvement in DNA repair processes and offering new insights into the expanding roles of CRISPR-Cas systems beyond adaptive immunity.

Many bacteria and archaea use CRISPR-Cas systems, which provide RNA-based, adaptive, and inheritable immune defenses against invading viruses and other foreign genetic elements. The proper processing of CRISPR guide RNAs (crRNAs) is a crucial step in the maturation of the defense complexes and is frequently performed by specialized ribonucleases encoded by cas genes. However, some systems employ enzymes associated with degradosome or housekeeping functions, such as RNase III or the endoribonuclease RNase E. Here, the endo- and 5′-exoribonuclease RNase J was identified as an additional enzyme involved in crRNA maturation, acting jointly with RNase E in the crRNA maturation of a type III-Bv CRISPR-Cas system, and possibly together with a further RNase in the cyanobacterium Synechocystis sp. PCC 6803. Co-IP experiments revealed a small set of proteins that were co-enriched with RNase J, among them the exoribonuclease polyribonucleotide nucleotidyltransferase (PNPase). Despite a measured, strong 3’ exonucleolytic activity of the recombinant enzyme, PNPase was not confirmed to contribute to crRNA maturation. However, the co-IP results indicate that PNPase in Synechocystis is an enzyme that can recruit either RNase E or RNase J, together with additional proteins.

Nlrp5 encodes a core component of the subcortical maternal complex (SCMC) a cytoplasmic protein structure unique to the mammalian oocyte and cleavage-stage embryo. NLRP5 mutations have been identified in patients presenting with early embryo arrest, recurrent molar pregnancies and imprinting disorders. Correct patterning of DNA methylation over imprinted domains during oogenesis is necessary for faithful imprinting of genes. It was previously shown that oocytes with mutation in the human SCMC gene KHDC3L had globally impaired methylation, indicating that integrity of the SCMC is essential for correct establishment of DNA methylation at imprinted regions. Here, we present a multi-omic analysis of an Nlrp5 - null mouse model, which in GV oocytes displays a misregulation of a broad range of maternal proteins, including proteins involved in several key developmental processes. This misregulation likely underlies impaired oocyte developmental competence. Amongst impacted proteins are several epigenetic modifiers, including a substantial reduction in DNMT3L; we show that de novo DNA methylation is attenuated in Nlrp5 -null oocytes. This provides evidence for mechanisms leading to downstream misregulation of imprinted genes, which in turn, may result in imprinting syndromes, multi-locus imprinting disturbances (MLID) and hydatidiform moles.

Summary Transitions between subsets of differentiating hematopoietic cells are widely regarded as unidirectional in vivo . Here, we introduce clonal phylogenetic tracer (CP-tracer) that sequentially introduces genetic barcodes, enabling high-resolution analysis of ∼100,000 subclones derived from ∼500 individual hematopoietic stem cells (HSC). This revealed previously uncharacterized HSC functional subsets and identified bidirectional fate transitions between myeloid-biased and lineage-balanced HSC. Contrary to the prevailing view that the more self-renewing My-HSCs unidirectionally transition to balanced-HSCs, phylogenetic tracing revealed durable lineage bidirectionality with the transition favoring My-HSC accumulation over time 1,2 . Further, balanced-HSCs mature through distinct intermediates—My-HSCs and lymphoid-biased-HSCs—with lymphoid competence here shown by CRISPR/Cas9 screening to be dependent on the homeobox gene, Hhex . Hhex enables Ly-HSC differentiation, but its expression declines with age. These findings establish HSC plasticity and Hhex as a determinant of myeloid-lymphoid balance with each changing over time to favor the age-related myeloid bias of the elderly. Highlights Sequenctial introduction of DNA barcodes in vivo was developed to assess time dependent changes in cell fate. Clonal phylogenetic tracer (CP-tracer) enabled high-resolution phylogenetic analysis of ∼100,000 subclones derived from ∼500 individual hematopoietic stem cells (HSC). Bidirectional fate transitions between myeloid-biased haematopoietic stem cells (My-HSCs) and lineage-balanced haematopoietic stem cells (balanced-HSCs) were observed. Hhex was identified as a molecular driver of HSC lymphoid competence.

ABSTRACT The transcription factor CCAAT/enhancer binding protein alpha (C/EBPα) regulates cell differentiation, proliferation, and function in various tissues, including the liver, adipose tissue, skin, lung, and hematopoietic system. Studies in rats, mice, humans, and chickens have shown that CEBPA mRNA undergoes alternative translation initiation, producing three C/EBPα protein isoforms. Two of these isoforms act as full-length transcription factors with N-terminal transactivation domains and a C-terminal dimerization and DNA-binding domains. The third isoform is an N-terminally truncated variant, translated from a downstream AUG codon. It competes with full-length isoforms for DNA binding, thereby antagonizing their activity. Expression of the truncated C/EBPα isoform depends on the initial translation of a short upstream open reading frame (uORF) in CEBPA mRNA and subsequent re-initiation at a downstream AUG codon, a process stimulated by mTORC1 signaling. We investigated whether the ortholog of the CEBPA gene in the evolutionarily distant, short-lived African turquoise killifish ( Nothobranchius furzeri ) is regulated by similar mechanisms. Our findings reveal that the uORF- mediated regulation of C/EBPα isoform expression is conserved in killifish. Disruption of the uORF selectively eliminates the truncated isoform, leading to unrestrained activity of the full-length C/EBPα isoforms. This genetic modification significantly extended both the median and maximal lifespan and improved the healthspan of male N. furzeri . These results highlight a conserved mechanism of CEBPA gene regulation across species and its potential role in modulating the lifespan and aging phenotypes.