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.

The codon usage bias of the Zika virus (ZIKV) genome is skewed towards AA-ending codons, which are preferentially decoded by U34-modified cognate tRNAs. This contrasts with the human host’s preference for AG-ending codons, suggesting that ZIKV may exploit specific tRNA modifications to optimize protein synthesis within human cells. To test this hypothesis, we used codon-biased eGFP sensors and found that ZIKV infection transiently increased the expression of AA-biased GFP at the expense of AG-biased GFP. Mass spectrometry analysis further showed that ZIKV virus infection increases mcm 5 s 2 U 34 tRNA modification content in host cells. In ELP1-deficient cells, which exhibit reduced U 34 modifications, ZIKV replication was impaired. Enhancing U 34 modification through using a small molecule known to restore ELP1 expression, rescued viral replication in these cells. Moreover, CRISPR/Cas9 and shRNA-mediated knockdown of key enzymes involved in U 34 modification, ELP1, ALKBH8, and CTU1, significantly reduced ZIKV replication. Collectively, these results provide strong evidence that ZIKV reprograms the host tRNA epitranscriptome and exploits host cell tRNA modifications, particularly at the wobble position U 34 , to optimize translation of its own proteins and promote viral replication.

Genomes assume a complex 3D architecture in the interphase cell nucleus. Yet, the molecular mechanisms that determine global genome architecture are only poorly understood. To identify mechanisms of higher order genome organization, we performed high-throughput imaging-based CRISPR knockout screens targeting 1064 genes encoding nuclear proteins in human cell lines. We assessed changes in the distribution of centromeres at single cell resolution as surrogate markers for global genome organization. The screens revealed multiple major regulators of spatial distribution of centromeres including components of the nucleolus, kinetochore, cohesins, condensins, and the nuclear pore complex. Alterations in centromere distribution required progression through the cell cycle and acute depletion of mitotic factors with distinct functions altered centromere distribution in the subsequent interphase. These results identify molecular determinants of spatial centromere organization, and they show that orderly progression through mitosis shapes interphase genome architecture.

The Greatwall kinase inhibits PP2A-B55 phosphatase activity during mitosis to stabilise critical Cdk1-driven mitotic phosphorylation. Although Greatwall represents a potential oncogene and prospective therapeutic target, our understanding of cellular and molecular consequences of chemical Greatwall inactivation remains limited. To address this, we introduce C-604, a highly selective Greatwall inhibitor, and characterise both immediate and long-term cellular responses to the chemical attenuation of Greatwall activity. We demonstrate that Greatwall inhibition causes systemic destabilisation of the mitotic phosphoproteome, premature mitotic exit and pleiotropic cellular pathologies. Importantly, we demonstrate that the cellular and molecular abnormalities linked to reduced Greatwall activity are specifically dependent on the B55α isoform rather than other B55 variants, underscoring PP2A-B55α phosphatases as key mediators of cytotoxic effects of Greatwall-targeting agents in human cells. Additionally, we show that sensitivity to Greatwall inhibition varies in different cell line models and that dependency on Greatwall activity reflects the balance between Greatwall and B55α expression levels. Our findings highlight Greatwall dependency as a cell-specific vulnerability and propose the B55α-to-Greatwall expression ratio as a predictive biomarker of cellular responses to Greatwall-targeted therapeutics.

Background Truncating variants in desmoplakin ( DSP tv), are a leading cause of arrhythmogenic cardiomyopathy (ACM), often presenting with early fibrosis and arrhythmias disproportionate to systolic dysfunction. DSP is critical for cardiac mechanical integrity, linking desmosomes to the cytoskeleton to withstand contractile forces. While loss-of-function is implicated, direct evidence, both for DSP haploinsufficiency in human hearts and for the impact of mechanical stress on cardiomyocyte adhesion, has been limited, leaving the pathogenic mechanism unclear. Methods We analyzed explanted human heart tissue from patients with DSP tv (N=3), titin truncating variants ( TTN tv, N=5), and controls (N=5) using RNA-sequencing and mass spectrometry. We generated human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) harboring patient-derived or CRISPR-Cas9 engineered DSP tv to model a range of DSP expression levels. Using a 2D cardiac muscle bundle (CMB) platform enabling live visualization of cell junctions, we developed an assay to assess cell-cell adhesion upon heightened contractile stress in response to the contractile agonist endothelin-1. CRISPR-interference (CRISPRi) was used to confirm the role of DSP loss, and CRISPR-activation (CRISPRa) was tested for therapeutic rescue. Results Compared to both control and TTN tv hearts, DSP tv human hearts exhibited reduced DSP at both the mRNA and protein level, as well as broadly disrupted desmosomal stoichiometry. Transcriptomic and proteomic analyses implicated cell adhesion, extracellular matrix, and inflammatory pathways. iPSC-CM models recapitulated DSP haploinsufficiency and desmosomal disruption. DSP tv CMBs showed normal baseline contractile function. However, they displayed marked cell-cell adhesion failure with contractile stress (75% failure vs. 8% in controls, p<0.001). Adhesion failure was prevented by the myosin inhibitor, mavacamten. CRISPRi-mediated DSP knockdown replicated this susceptibility to adhesion failure. Conversely, CRISPRa robustly increased DSP expression and rescued cell-cell adhesion failure in DSP tv CMBs (9% failure post-CRISPRa, p<0.001 vs. un-treated). Rescue occurred even when only the DSPII isoform was upregulated in a model with biallelic DSP transcript 1 loss of function. Conclusions DSP haploinsufficiency is the major cause of DSP cardiomyopathy with a primary consequence of conferring vulnerability to cardiomyocyte cell-cell adhesion failure under heightened contractile stress. Transcriptional activation of DSP reverses this defect in preclinical models, establishing proof-of-concept for a potential therapeutic strategy in DSP cardiomyopathy.

Bovine Parainfluenza Virus Type 3 (BPIV3) is a leading cause of respiratory illness in cattle and a primary component of Bovine Respiratory Disease Complex (BRDC), resulting in significant economic losses. Understanding the mechanisms of BPIV3 infection, particularly the entry process, is essential for developing effective control measures. Identifying specific host factors that viruses exploit during their life cycle can reveal critical vulnerabilities for potential antiviral targets. We established a genome-wide CRISPR/Cas9 knockout screen in bovine cells to identify host factors involved in viral infections. Our screen identified several key host factors required for BPIV3 infection, including the sialic acid transporter SLC35A1 and the protein LSM12. Further mechanistic analysis revealed that these factors play critical roles at distinct stages of the BPIV3 entry process. These findings not only advance our understanding of how BPIV3 infects host cells, but also identify potential host targets for inhibiting infection and developing novel antiviral strategies.

Abstract The yeast Saccharomyces cerevisiae is widely employed in industrial biotechnology for chemical and pharmaceutical production. However, engineering yeast for high product titers remains challenging due to metabolic imbalances and competition for cellular resources. To address this, we developed an orthogonal quorum sensing (QS) system based on N -acyl-homoserine lactones (AHLs) for cell density-dependent regulation in yeast. Using metabolic engineering, we established AHL production in yeast. Next, we improved AHL-biosensors via directed evolution and a novel growth-based screening strategy with amdS as a counter-selectable marker. We identified LuxR variants with enhanced sensitivity, which were engineered for QS-controlled expression of a reporter gene. Additionally, we engineered LuxR to function as a repressor, achieving QS-dependent repression. The QS system was applied to enhance aloesone production, a plant-derived metabolite with cosmetic and pharmaceutical applications. The established system showed 51% increased production through QS-controlled repression of FAS1 . This work establishes a versatile QS-based regulatory platform to support dynamic pathway regulation for metabolic engineering in yeast.

Abstract Stargardt’s Disease (STGD1), Retinitis pigmentosa (RP), and Leber’s congenital amaurosis (LCA) inherited eye conditions that are among the main causes of blindness and visual impairment. The primary cause of these diseases, which lead to gradual vision loss, is genetic mutations affecting retinal cells. Gene-editing technologies, especially the CRISPR-Cas9 system, are emerging as promising ways to correct harmful genetic mutations. This review focuses on how effectively CRISPR-Cas9 can be used to treat certain inherited eye conditions. In experiments with patient-derived human induced pluripotent stem cells (hiPSCs), researchers were able to fix STGD1 mutations in the ABCA4 gene using CRISPR-Cas9. The corrected cells retained their ability to differentiate into different cell types and showed minimal off-target effects. This suggests the approach could offer a safe and precise way of treating these conditions. In RP, CRISPR-Cas9 was used to target the RPGR gene in mice models, successfully preserving the shape and function of the photoreceptor cells. The CEP290 mutation in LCA10 was corrected in human patients using the CRISPR-Cas9 based EDIT-101 in the BRILLIANCE clinical trial, which demonstrated a notable improvement in visual outcomes with minimal adverse effects. Even though the results so far are promising, challenges remain including long-term safety, the administration of these treatments, and the risk of unintended effects. This review emphasizes the need for further research and clinical trials to improve these therapies. It also highlights the exciting potential of CRISPR-Cas9 as a curative treatment for inherited retinal disorders.

ABSTRACT Mutations in the dystrophin (DMD) gene can cause a spectrum of muscle-wasting disorders ranging from the milder Becker muscular dystrophy (BMD) to the more severe Duchenne muscular dystrophy (DMD). Among these, exon 45 deletion is the most frequently reported single exon deletion in DMD patients worldwide. In this study, we generated a novel rat model with an exon 45 deletion using CRISPR/Cas9 technology. The Dmd Δ45 rat recapitulate key clinical and molecular features of DMD, including progressive skeletal muscle degeneration, cardiac dysfunction, cognitive deficits, elevated circulating muscle damage biomarkers, impaired muscle function, and overall reduced lifespan. Transcriptomics analyses confirmed the deletion of exon 45 and revealed gene expression patterns consistent with dystrophin deficiency. In the skeletal muscle, RNA-seq profiles demonstrated a transition from early stress responses and regenerative activity at 6 months to chronic inflammation, fibrosis, and metabolic dysfunction by 12 months. Similarly, the cardiac transcriptomic shifted from an early inflammatory and stress-responsive state to one characterized by fibrotic remodelling and metabolic impairment. Despite these pathological features, the Dmd Δ45 rats exhibited a milder phenotype than other DMD rat models. This attenuation may be attributed to spontaneous exon 44 skipping, which partially restores the reading frame and results in an age-dependent increase in revertant dystrophin-positive fibres. Further analysis indicated downregulation of spliceosome-related genes, suggesting a potential mechanism driving exon skipping in this model. In summary, the Dmd Δ45 rat represents a valuable model for investigating both the molecular determinants of phenotypic variability and the endogenous mechanisms of exon skipping. These findings offer important insights for the development of personalized exon-skipping therapies, particularly for DMD patients with exon 45 deletions.

A GGGGCC repeat expansion in C9orf72 is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). The repeat expansion is translated into five different dipeptide repeat proteins: polyGA, polyGP, polyGR, polyAP and polyPR. To investigate the effect of polyGA, which is the most abundant dipeptide repeat protein in patient brains, we used CRISPR/Cas9 to insert 400 codon-optimized polyGA repeats immediately downstream of the mouse C9orf72 start codon. This generated (GA)400 knock-in mice driven by the endogenous mouse C9orf72 promoter, coupled with heterozygous C9orf72 reduction. (GA)400 mice develop subtle pathology including mild motor dysfunction characterized by impaired rotarod performance. Quantitative proteomics revealed polyGA expression caused protein alterations in the spinal cord, including changes in previously identified polyGA interactors. Our findings show that (GA)400 mice are a complementary in vivo model to better understand C9ALS/FTD pathology and determine the specific role of single DPRs in disease.