Genome-wide association studies (GWAS) have identified the SAMM50 rs3761472 single nucleotide polymorphism (SNP) as a risk factor for metabolic dysfunction-associated steatotic liver disease (MASLD), although its in vivo functions remain unclear. SAMM50 encodes a mitochondrial outer membrane protein critical for maintaining mitochondrial structure. To investigate the biological effects of rs3761472, we generated Samm50 -knock-in (KI) mice harboring a D110G substitution using CRISPR/Cas9. This variant impaired mitochondrial integrity by downregulating key regulators of mitochondrial architecture, dynamics, and quality control. This contributed to reduced ATP production and elevated oxidative stress, inflammation, and hepatocyte death. The mutation also induced insulin resistance and glucose intolerance. Samm50 -KI mice fed a high-fat diet exhibited pronounced hepatic lipid accumulation and liver injury, highlighting its pathogenic role in MASLD progression. Our findings demonstrate that SAMM50 rs3761472 is a critical driver of mitochondrial dysfunction and MASLD susceptibility, supporting its potential as a therapeutic target and its relevance to precision medicine.

ABSTRACT Serum response factor (SRF) and its cofactors, Myocardin-related transcription factors A/B (MRTF-A/B), regulate transcription of numerous cytoskeletal structural and regulatory genes, and most MRTF/SRF inactivation phenotypes reflect deficits in cytoskeletal dynamics. We show that MRTF-SRF activity is required for effective proliferation of both primary and immortalised fibroblast and epithelial cells. Cells lacking the MRTFs or SRF proliferate very slowly, express elevated levels of SASP factors and SA-β-galactosidase activity, and inhibit proliferation of co-cultured primary wildtype cells. They exhibit decreased levels of CDK1 and CKS2 proteins, and elevated levels of CDK inhibitors, usually CDKN1B/p27. These phenotypes, which can be fully reversed by re-expression of MRTF-A, are also seen in wildtype cells arrested by serum deprivation. Moreover, in wildtype cells direct interference with cytoskeletal dynamics through inhibition of ROCKs or Myosin ATPase induces a similar proliferative defect to that seen in MRTF-null cells. MRTF-null cells exhibit multiple cytoskeletal defects, and markedly reduced contractility. We propose that MRTF-SRF driven cytoskeletal dynamics and contractility are essential for operation of the pro-proliferative signal provided by cell-substrate adhesion.

ABSTRACT Essential genes are interesting in their own right and as potential antibiotic targets. To date, only one report has identified essential genes on a genome-wide scale in Clostridioides difficile , a problematic pathogen for which treatment options are limited. That foundational study used large-scale transposon mutagenesis to identify 404 protein-encoding genes as likely to be essential for vegetative growth of the epidemic strain R20291. Here, we revisit the essential genes of strain R20291 using a combination of CRISPR interference (CRISPRi) and transposon-sequencing (Tn-seq). First, we targeted 181 of the 404 putatively essential genes with CRISPRi. We confirmed essentiality for >90% of the targeted genes and observed morphological defects for >80% of them. Second, we conducted a new Tn-seq analysis, which identified 346 genes as essential, of which 283 are in common with the previous report and might be considered a provisional essential gene set that minimizes false positives. We compare the list of essential genes to those of other bacteria, especially Bacillus subtilis , highlighting some noteworthy differences. Finally, we used fusions to red fluorescent protein (RFP) to identify 18 putative new cell division proteins, three of which are conserved in Bacillota but of largely unknown function. Collectively, our findings provide new tools and insights that advance our understanding of C. difficile . IMPORTANCE Clostridioides difficile is an opportunistic pathogen for which better antibiotics are sorely needed. Most antibiotics target pathways that are essential for viability. Here we use saturation transposon mutagenesis and gene silencing with CRISPR interference to identify and characterize genes required for growth on laboratory media. Comparison to the model organism B. subtilis reveals many similarities and a few striking differences that warrant further study and may include opportunities for developing antibiotics that kill C. difficile without decimating the healthy microbiota needed to keep C. difficile in check.

ABSTRACT LIMD1 (LIM domains containing 1) is a bona-fide tumour suppressor gene frequently lost during the early stages of non-small cell lung cancer (NSCLC) development, resulting in a worse outcome for LIMD1 -deficient patients. LIMD1 deficiency is present in approximately 50% of NSCLC cases, representing at least 21,000 patients in the United Kingdom and 1.2 million worldwide. During cancer progression, cells can accumulate genetic changes that render them more dependent on certain genes for their survival. By performing CRISPR-Cas9 dropout screens, we identified GPX4 as a novel vulnerability in LIMD1 deficient LUAD cells. By targeting GPX4 using RNA interference (RNAi) and pharmacological intervention in our isogenic LUAD lines along with a non-transformed lung cell line, we validated GPX4 as a novel dependency in LIMD1 deficient cells. GPX4 is a key defence mechanism against ferroptosis, an iron-dependent form of regulated cell death. This state of increased ferroptosis susceptibility upon LIMD1 loss is due to increased basal reactive oxygen species levels and lipid peroxidation. Importantly, targeting GPX4 with ferroptosis inducing agents in NSCLC patients with LIMD1 loss may represent a novel therapeutic strategy.

Summary The influenza A virus polymerase, consisting of a heterotrimer of three viral proteins, carries out both transcription and replication of the viral RNA genome. These distinct activities are regulated by viral proteins that vary in abundance during infection, and by various co-opted host cell proteins, which serve as targets for the development of novel antiviral interventions. However, little is known about which host proteins direct transcription and which replication. In this report, we performed a differential interactome screen to identify host proteins co-opted as either transcription-or replication-specific factors. We found that distinct sets of host proteins interact with the influenza polymerase as it carries out the different activities. We functionally characterised HMGB2 and RUVBL2 as replication-specific cofactors and RPAP2 as a transcription-specific cofactor. Our data demonstrate that comparative proteomics can be used as a targeted approach to uncover virus-host interactions that regulate specific stages of the viral lifecycle.

The planthopper Pentastiridius leporinus has emerged as a severe crop pest, rapidly expanding both its host plant range and the affected areas in central Europe. Originating as a monophagous herbivore of reed grass, P. leporinus recently adopted polyphagous feeding and is now a pest of sugar beet, potato, carrot, and onion, suggesting rapid ecological niche expansion. P. leporinus vectors two bacterial pathogens, the γ-proteobacterium Candidatus Arsenophonus phytopathogenicus (CAP) and the stolbur phytoplasma Candidatus Phytoplasma solani (CPS), which are responsible for a range of disease syndromes, including syndrome basses richesses (SBR) in sugar beet. We used long-read metagenomic sequencing to characterize the genomes of microbes associated with P. leporinus , resulting in the complete sequences of CAP and CPS, as well as primary symbionts of the genera Purcelliella, Sulcia and Vidania , and facultative symbionts Rickettsia and Wolbachia . The primary symbionts are inferred to provide all ten essential amino acids and contribute to B vitamin biosynthesis. The genomes of CPS and CAP encode numerous pathogenicity factors, enabling the colonization of different hosts. Bacterial fluorescence in situ hybridization revealed the tissue distribution, cellular localization, relative abundance and transmission patterns of these bacteria. The intracellular presence of all primary symbionts in bacteriomes, the intracellular presence of Wolbachia , and the intranuclear localization of Rickettsia , suggest vertical transmission. CPS was restricted to salivary glands, suggesting strict horizontal, plant-mediated transmission, whereas CAP colonized all tissue types, allowing for horizontal and vertical transmission. Our data suggest that P. leporinus hosts an exceptionally broad range of symbionts, encompassing mutualistic, commensal and pathogenic interactions. Importance The planthopper Pentastiridius leporinus has recently expanded its host plant range and emerged as severe pest of sugar beet and potato crops in central Europe, which is exacerbated by its capacity to vector bacterial pathogens to its host plants. Because microbial symbionts may play an important role for both the host plant shifts and the transmission of pathogens, we used metagenomic sequencing and fluorescence in situ hybridization to characterize the microbial community associated with P. leporinus . We detected three bacteriome-localized primary symbionts that together provision all essential amino acids and several B-vitamins to the host, as well as two intracellular bacteria with a broader tissue distribution. In addition, we infer localization, transmission, and putative pathogenicity factors for the two major phytopathogens that are vectored by P. leporinus . Our results reveal a complex community of symbiotic bacteria that likely shapes the interaction of this emerging pest with its host plants.

Abstract While genetic screens have facilitated the dissection of protein function in animal development, advances in systematic point mutagenesis open new opportunities for forward genetics in mammalian cells. Here, we develop a CRISPR/Cas9-mediated base editing screen that allows to generate extensive maps of single amino acid substitutions of endogenous proteins. We demonstrate the application on the X-chromosomal Hprt and autosomal Msh2 gene in diploid male and haploid mouse embryonic stem cells, respectively. Finally, we use this methodology to generate a sequence-function map of the transcriptional co-repressor SPEN in X chromosome inactivation. We demonstrate that the substitution of the SPEN RRM4-residue W522 abrogates X-linked gene repression by Xist RNA and impairs H3K27-deacetylation, but does not affect Polycomb recruitment and H3K27me3 deposition. Our results demonstrate that screening in haploid cells allow the efficient identification of separation-of-function mutations that would be recessive in diploid cells suggesting applications to a wide range of areas

Abstract Antimicrobial resistance (AMR) is a critical global health threat, and artificial intelligence (AI) presents new opportunities to combat it. However, research priorities at the AI-AMR intersection remain undefined. This study aimed to identify and prioritise key areas for future investigation. Using a modified James Lind Alliance approach, we conducted semi-structured interviews with eight experts in AI and AMR between February and June 2024. Analysis of 338 coded responses revealed 44 distinct themes. Major barriers included fragmented data access, integration challenges, and economic disincentives. The top ten priorities identified were: Combination Therapy, Novel Therapeutics, Data Acquisition, AMR Public Health Policy, Prioritisation, Economic Resource Allocation, Diagnostics, Modelling Microbial Evolution, AMR Prediction, and Surveillance. A notable limitation was the underrepresentation of data from high-burden regions, affecting model generalisability. To address these gaps, we propose the novel BARDI framework: Brokered Data-sharing, AI-driven Modelling, Rapid Diagnostics, Drug Discovery, and Integrated Economic Prevention.

Abstract Aberrant DNA methylation of tumor-suppressive microRNAs is a frequent epigenetic alteration driving cancer progression. This study explores the targeted demethylation of the miR-200c promoter, a key regulator of epithelial–mesenchymal transition (EMT), using the CRISPR/dCas9-TET1 system in breast cancer cell lines. Two specific sgRNAs were designed to guide the dCas9-TET1 construct to the miR-200c promoter in MCF-7 and MDA-MB-231 cells. High-Resolution Melting (HRM) analysis showed decreased promoter methylation around 50% in MDA-MB-231 and moderately in MCF-7. Quantitative PCR confirmed a significant increase in miR-200c expression, with combined sgRNAs producing a synergistic effect in MCF-7, while only sgRNA1 was effective in MDA-MB-231. The increased expression of miR-200c led to downregulation of its targets ZEB1, ZEB2, and KRAS. Functionally, demethylation reduced cell proliferation and induced apoptosis, as shown by MTT assays and apoptosis analysis. These findings suggest that CRISPR/dCas9-TET1-mediated epigenetic editing can reactivate silenced miR-200c and inhibit malignant traits in breast cancer cells. This approach highlights a promising therapeutic strategy targeting the epigenome to restore tumor-suppressive functions.

Microglia dynamically support brain homeostasis through the induction of specialized activation programs or states. One such program is the Interferon-Responsive Microglia state (IRM), which has been identified in developmental windows, aging, and disease. While the functional importance of this state is becoming increasingly clear, our understanding of the regulatory networks that govern IRM induction remain incomplete. To systematically identify genetic regulators of the IRM state, we conducted a genome-wide CRISPR interference (CRISPRi) screen in human iPSC-derived microglia (iPS-Microglia) using IFIT1 as a representative IRM marker. We identified 772 genes that modulate IRM, including canonical type I interferon signaling genes ( IFNAR2, TYK2, STAT1/2, USP18 ) and novel regulators. We uncovered a non-canonical role for the CCR4-NOT complex subunit CNOT10 in IRM activation, independent of its traditional function. This work provides a comprehensive resource for dissecting IRM biology and highlights both established and novel targets for modulating microglial interferon signaling in health and disease.

Type I-E CRISPR-Cas3 derived from Escherichia coli (Eco CRISPR-Cas3) can introduce large deletions in target sites and is available for mammalian genome editing. The use of Eco CRISPR-Cas3 to plants is challenging because 7 CRISPR-Cas3 components (6 Cas proteins and CRISPR RNA) must be expressed simultaneously in plant cells. To date, application has been limited to maize protoplasts, and no mutant plants have been produced. In this study, we developed a genome editing system in rice using Eco CRISPR-Cas3 via Agrobacterium -mediated transformation. Deletions in the target gene were detected in 39–48% and 55–71% of calli transformed with 2 binary vectors carrying 7 expression cassettes of Eco CRISPR-Cas3 components and a compact all-in-one vector carrying 3 expression cassettes of Cas proteins fused to 2A self-cleavage peptide, respectively. The frequency of alleles lacking a region 7.0 kb upstream of the PAM sequence was estimated as 21–61% by quantifying copy number by droplet digital PCR, suggesting that mutant plants could be obtained with reasonably high frequency. Deletions were determined in plants regenerated from transformed calli and stably inherited to the progenies. Sequencing analysis showed that deletions of 0.1–7.2 kb were obtained, as reported previously in mammals. Interestingly, deletions separated by intervening fragments or with short insertion and inversion were also determined, suggesting the creation of novel alleles. Overall, Eco CRISPR-Cas3 could be a promising genome editing tool for gene knockout, gene deletion, and genome rearrangement in plants.

ABSTRACT The biogenesis of thousands of highly diverse membrane proteins in humans is facilitated by an array of ER-resident membrane protein translocases. While some membrane proteins have a strict requirement for a specific insertion machinery, membrane proteins with short translocated domains may be able to access multiple pathways. Here, we quantify the functional importance of redundancy in membrane protein translocation during influenza A virus (IAV) infection by examining the biogenesis of the viroporin M2. Given the wide host and cellular tropism of IAV, the virus likely evolved mechanisms to leverage host translocation pathways efficiently. We demonstrate that although M2 utilizes the ER membrane protein complex (EMC), driven by signals encoded in its transmembrane and C-terminal domains, M2 maintains an approximately 50% membrane insertion rate in the absence of the EMC. This influences viral cell-to-cell transmission across different IAV strains, with a greater impact on those expressing lower levels of M2. We identify alternative translocation of M2 via Oxa1-family translocons independent of canonical targeting chaperones. These findings reveal how the exploitation of multiple redundant pathways can ensure robust IAV infection. SIGNIFICANCE STATEMENT IAV must rapidly replicate in diverse mammalian hosts, which requires efficient integration of viral proteins into host cell membranes. This study uncovers how the viral proton channel M2 utilizes multiple redundant protein insertion pathways, accessing EMC and alternative Oxa1-family translocases. Revealing these redundant strategies clarifies how cells triage membrane proteins, offering insights into both viral adaptation and host cell robustness.

The receptor-like cytoplasmic kinase BIK1 and its close homolog PBL1 have been widely recognized as central components of plant immunity. However, most genetic studies of BIK1 and PBL1 functions were carried out with single T-DNA insertional mutant alleles. Some phenotypes observed in these mutants, e.g. autoimmunity, have been difficult to reconcile with the proposed role of BIK1 and PBL1 in pattern-triggered immunity. In this study, we generated multiple new alleles of bik1 and pbl1 by CRISPR-Cas9-based gene editing and systematically analyzed these mutants alongside existing T-DNA insertional lines. These analyses reinforced the central role of BIK1 and PBL1 in pattern-triggered immunity mediated by both receptor kinases and receptor-like proteins. At the same time, however, we revealed several pleiotropic phenotypes associated with T-DNA insertions that are not necessarily linked to loss of BIK1 or PBL1 function. Further analyses of newly generated bik1 pbl1 double mutants uncovered an even greater contribution of these kinases to immune signaling and disease resistance than previously appreciated. These findings clarify longstanding ambiguities surrounding BIK1 and PBL1 functions.

Soybean ( Glycine max ) has not yet benefited from large-scale hybrid breeding efforts due to its small, self-fertilizing flowers that are difficult to emasculate, and limited attractiveness to pollinators. This study explores targeted floral trait engineering to enhance pollinator attraction, aiming to overcome barriers to soybean hybridization. We generated a high-resolution floral organ expression atlas and H3K4 trimethylation ChIP-Seq dataset to identify candidate genes involved in petal development, nectar sugar content, and petal pigmentation. Using CRISPR-based activation and repression systems, we modified the expression of AINTEGUMENTA (GmANT) , BIGPETAL (GmBPE) , and SUCROSE TRANSPORTER2 (GmSUC2) . Contrary to expectations based on Arabidopsis homologs, transcriptional activation of GmANT_B and repression of GmBPE led to reduced, rather than increased, petal size, highlighting divergent regulatory mechanisms in soybean. Complementation of the W1 gene that controls petal pigmentation successfully converted white petals to purple, with preliminary evidence indicating that this color conversion may increase pollinator visitation. These results underscore the complexity of floral development in soybean and provide foundational tools and resources for future efforts to engineer reproductive traits for hybrid seed production.

Rice ( Oryza sativa L.) is a staple food for half of the world’s population, but lacks essential nutrients such as iron (Fe). Fe deficiency is one of the most common nutritional problems in humans, and biofortification of rice grains is a cost-effective approach to deliver more Fe to people’s diet. Two Vacuolar Iron Transporters, OsVIT1 and OsVIT2, were shown to negatively regulate Fe translocation to rice developing panicles, as single mutants osvit1 and osvit2 have increased Fe concentration in seeds. Importantly, rice plants are frequently cultivated in waterlogged soils that are highly reductive and prone to Fe 3+ reduction to the more soluble Fe 2+ , which can accumulate and cause Fe toxicity. Little is known about which genes control Fe excess detoxification. OsVIT1 and OsVIT2 transport Fe into the vacuole, and OsVIT2 is induced under Fe excess, but whether they play a role in Fe detoxification was not demonstrated. We generated double mutants osvit1osvit2 using CRISPR-Cas9 to test whether loss of function of both genes could increase Fe concentration in seeds, and to test whether their loss of function has impact in rice Fe excess tolerance. We showed that osvit1osvit2 double mutants accumulated more Fe in brown rice. Fe accumulation was clear in embryo scutellum and plumule, suggesting VIT transporters have a role in determining Fe spatial distribution. We also showed that root uptake contributed significantly for increased Fe accumulation in osvit1osvit2 seeds, suggesting OsVIT1 and OsVIT2 are involved in sequestering Fe in vegetative tissues and decreasing translocation. Strikingly, we found that osvit1osvit2 plants were more sensitive to Fe excess, revealing a trade-off between Fe biofortification and Fe excess tolerance. Our data indicates OsVIT1 and OsVIT2 are key for Fe excess detoxification, which should be considered in their use as targets for biofortification.

Cas12a (Cpf1) is a class 2 CRISPR-Cas effector protein with RNA-guided DNA endonuclease activity widely used for genome editing. While its DNA cleavage and target recognition mechanisms have been studied extensively, the possibility of auxiliary enzymatic functions remains underexplored. Here, I report that Acidaminococcus sp. Cas12a (AsCas12a) possesses intrinsic ATPase activity, despite lacking canonical nucleotide-binding or hydrolysis motifs. Using a radiometric thin-layer chromatography (TLC) assay, I demonstrate that AsCas12a hydrolyzes ATP in a concentration and time-dependent manner. Importantly, this activity occurs independently of DNA cofactors, as neither single-stranded nor double-stranded DNA influenced the rate or extent of ATP hydrolysis. Bioinformatic analyses using NsitePred and SwissDock identified potential ATP-binding residues with predicted favorable binding energies. This preliminary finding uncovers a previously unrecognized biochemical property of AsCas12a and raises questions regarding the physiological role of this ATPase activity in CRISPR function.

SUMMARY Gene expression shapes the nervous system at every biological level, from molecular and cellular processes defining neuronal identity and function to systems-level wiring and circuit dynamics underlying behaviour. Here, we generate the first high-resolution, single-cell transcriptomic atlas of the adult Drosophila melanogaster central brain by integrating multiple datasets, achieving an unprecedented tenfold coverage of every neuron in this complex tissue. We show that a neuron’s genetic identity overwhelmingly reflects its developmental origin, preserving a genetic address based on both lineage and birth order. We reveal foundational rules linking neurogenesis to transcriptional identity and provide a framework for systematically defining neuronal types. This atlas provides a powerful resource for mapping the cellular substrates of behaviour by integrating annotations of hemilineage, cell types/subtypes and molecular signatures of underlying physiological properties. It lays the groundwork for a long-sought bridge between developmental processes and the functional circuits that give rise to behaviour.

Summary The insulin receptor (INSR) exists in two isoforms, INSR-A and INSR-B, resulting from alternative splicing of the INSR pre-mRNA. INSR-B mediates the metabolic and mitogenic effects of insulin in the adult liver, while INSR-A is expressed during development. Recently, INSR-A has been detected in pathological murine and human livers. Here, we develop an in vivo CRISPR/Cas9 strategy to assess the impact of INSR-A on mouse liver homeostasis and susceptibility to carcinogenesis. We find that INSR-A expression in hepatocytes leads to the spontaneous development of liver tumours and also increases tumour initiation in a context of β-catenin-driven liver carcinogenesis. Mechanistically, this is attributed to the higher intrinsic capacity of INSR-A expressing hepatocytes to enter apoptosis, rendering the microenvironment more inflammatory, thus making way for the proliferation of preneoplastic cells. Collectively, our data highlight a novel function for INSR-A in promoting liver cancer via non-cell autonomous mechanisms.

ARC syndrome (arthrogryposis-renal dysfunction-cholestasis) is a rare autosomal recessive multisystem disorder affecting the kidneys. The disease is caused by mutations in either VPS33B or VIPAS39 . ARC syndrome is currently incurable, with patients rarely surviving beyond their first year of life. The renal component of this disorder is characterised by proximal tubular dysfunction. Here, a proximal tubular cell line, RPTEC-TERT1, was CRISPR-edited to knock out (KO) VPS33B expression. Characterisation of VPS33B -KO cells was performed using brightfield imaging, immunostaining, RNA sequencing, and cell detachment assays. The VPS33B -KO RPTEC-TERT1 cells demonstrated a ‘peeling’ phenotype and altered cell adhesion. This, along with altered transcription of genes associated with cell adhesion, suggests that VPS33B KO results in cell-matrix attachment defects. These findings provide the first insights into the cause of proximal tubular dysfunction in ARC syndrome.

SUMMARY Sex differences in behaviours arise from variations in female and male nervous systems, yet the cellular and molecular bases of these differences remain poorly defined. Here, we take an unbiased, single-cell transcriptomic approach to uncover how sex shapes the adult Drosophila melanogaster brain. We show that sex differences do not result from large-scale transcriptional reprogramming but through fine-tuning of otherwise shared developmental templates via the sex-differentiating transcription factors Doublesex and Fruitless. We reveal, with unprecedented resolution, the extraordinary genetic diversity within these sexually dimorphic cell types and find birth order represents a novel axis of sexual differentiation. Neuronal identity in the adult reflects spatiotemporal patterning and sex-specific survival, with female-biased neurons arising early and male-biased neurons arising late. This pattern reframes dimorphic neurons as “paralogous” rather than “orthologous”, suggesting sex leverages distinct developmental windows to build behavioural circuits and highlights a role for exaptation in diversifying the brain.

Abstract Background Long Interspersed Nuclear Elements-1 (LINE-1, L1) are transposable elements that make up roughly 17% of the human genome. These elements can copy and insert themselves into new genomic locations [1]. Typically, LINE-1 is repressed in healthy tissues but may become activated in various human diseases. LINE-1 expression has been associated with aging [2–4], neurodegenerative disorders [5–7], cancer [8–10], and autoimmune diseases [11,12]. Despite the strong association between LINE-1 expression and disease, the regulatory mechanisms controlling the expression of LINE-1-encoded ORF1p and ORF2p and the link between LINE-1 activity and cancer cell survival remain poorly understood. Elucidating these mechanisms will deepen our understanding of how LINE-1 contributes to disease pathogenesis. Results To identify upstream regulators of LINE-1 and genes associated with LINE-1 activity-dependent lethality, we developed a dual-reporter system that simultaneously monitors the protein levels of LINE-1-encoded ORF1p and ORF2p (wild-type or catalytically inactive EN/RT mutant). Using genome-wide CRISPR/Cas9-based screens with this reporter system, we identified genes that control LINE-1 expression through multiple potential mechanisms, including their regulation at both RNA and protein levels. Besides known regulators like the HUSH complex, our screening uncovered previously unknown regulators of ORF1p and ORF2p, and revealed distinct mechanisms regulating expression of these proteins. We also identified genes whose disruption contributes to LINE-1 activity-dependent lethality. Conclusion This study offers a valuable resource for the retrotransposon field, providing new insights into the distinct molecular mechanisms regulating LINE-1-encoded ORF1p and ORF2p, and highlighting potential therapeutic targets for diseases driven by LINE-1 dysregulation.

Abstract Animal models of human disease syndromes are useful for elucidating disease causes and developing treatments. The protocadherin γC4 (Pcdh-γC4) among the 22 isoforms encoded by the protocadherin-γ (Pcdh-γ) gene cluster causes a human neurodevelopmental syndrome with progressive microcephaly, seizures, and intellectual disorder. Here, we successfully established a useful mouse model of human Pcdh-γC4 neurodevelopmental syndrome, which exhibited motor disfunctions, seizures, brain size reduction, massive neuronal apoptosis and impaired dendritic development of Purkinje cells. At the same times, we generated Pcdh-γC4 only mice that express only full-length Pcdh-γC4 and truncated forms of the other 21 Pcdh-γ isoforms by using a two-step CRISPR/Cas9-based genome editing strategy, DOMINO (Double Mutation Inducing Open reading frame switch). These mice were viable and fertile, unlike Pcdh-γ cluster knockout mice. Biochemical analyses revealed that Pcdh-γC4 significantly regulates phosphorylation of FAK and PYK2, implicating its γ-constant domain in intracellular signaling. Furthermore, we found that Pcdh-γC4 is sufficient to restore normal dendritic self-avoidance of Purkinje cells observed in Pcdh-γ full-cluster knockouts. These findings demonstrate that Pcdh-γC4 is both necessary and sufficient for neuronal survival, dendritic patterning, and signaling, highlighting it as a functionally dominant isoform within the Pcdh-γ gene cluster.

This work explores the application of yeast in beer brewing and winemaking, comparing traditional genetic manipulation methods with innovative techniques, both GMO-free and GMO-based. Traditional approaches, such as sexual breeding and random mutagenesis, are contrasted with modern methods like Adaptive Laboratory Evolution (ALE), the integration of big data, AI, and omics technologies, and synthetic microbial communities, which do not involve genetic modification. GMO-based techniques, including synthetic biology and CRISPR/Cas9, enable more precise and efficient genome modifications, making multiplex genome engineering scalable, thanks to the efficient recombination machinery of Saccharomyces cerevisiae. Despite the promise of these advanced techniques, the commercialization of GMO-based methods faces significant challenges.

As the prevalence of age-related diseases rises, understanding and modulating the aging process is becoming a priority. Transcriptomic aging clocks (TACs) hold great promise for this endeavor, yet most are hampered by platform or tissue specificity and limited accessibility. Here, we introduce Pasta, a robust and broadly applicable TAC based on a novel age-shift learning strategy. Pasta accurately predicts relative age from bulk, single-cell, and microarray data, capturing senescent and stem-like cellular states through signatures enriched in p53 and DNA damage response pathways. Its predictions correlate with tumor grade and patient survival, underscoring clinical relevance. Applied to the CMAP L1000 dataset, Pasta identified known and novel age-modulatory compounds and genetic perturbations, and highlighted mitochondrial translation and mRNA splicing as key determinants of the cellular propensity for aging and rejuvenation, respectively. Supporting Pasta’s predictive power, we validated pralatrexate as a potent senescence inducer and piperlongumine as a rejuvenating agent. Strikingly, chemotherapy drugs were highly enriched among pro-aging hits. Taken together, Pasta represents a powerful and generalizable tool for aging research and therapeutic discovery, distributed as an easy-to-use R package on GitHub.

In mitosis the duplicated genome is aligned and accurately segregated between daughter nuclei. CTCF is a chromatin looping protein in interphase with an unknown role in mitosis. We previously published data showing that CTCF constitutive knockdown causes mitotic failure, but the mechanism remains unknown. To determine the role of CTCF in mitosis, we used a CRISPR CTCF auxin inducible degron cell line for rapid degradation. CTCF degradation for 3 days resulted in increased failure of mitosis and decreased circularity in post-mitotic nuclei. Upon CTCF degradation CENP-E is still recruited to the kinetochore and there is a low incidence of polar chromosomes which occur upon CENP-E inhibition. Instead, immunofluorescence imaging of mitotic spindles reveals that CTCF degradation causes increased intercentromere distances and a wider and more disorganized metaphase plate, a disruption of key functions of the pericentromere. These results are similar to partial loss of cohesin, an established component of the pericentromere. Thus, we reveal that CTCF is a key maintenance factor of pericentromere function, successful mitosis, and post-mitotic nuclear shape.