Abstract In wheat, inflorescence architecture critically determines yield potential, yet its structural complexity and asynchronous development have hindered cellular-resolution studies of spikelet and floret formation. Here, we integrate spatial transcriptomics, high-sensitivity multiplexed error-robust fluorescence in situ hybridization (MERFISH), and snRNA-seq across six developmental stages to generate a spatiotemporal atlas of the wheat inflorescence. We identified 20 cell types, spatially resolved into three categories: 1) proliferating cells within spikelet, marked by active division; 2) supporting cells along the central axis, including pith, cortex, and vasculature; and 3) developmental cells located both inside and at the base of the spikelets. The multi-omics approach enabled identification of the rare cell type ovary. Trajectory inference revealed that spikelets and florets originate from two temporally and spatially distinct sub-clusters of proliferating cells (R7), each defined by high expression of developmental regulators. These findings challenge the conventional model sequential meristem transitions (inflorescence-spikelet-floret) in wheat. Integration of time-series snATAC-seq and snRNA-seq delineated cellular transcriptional regulatory networks (cTRNs) governing spikelet formation, mediated by auxin and cytokinin signaling, and floret formation, driven by MADS-box transcription factors. Cell identity was maintained by cell type-specific accessible chromatin regions (csACRs), which are enriched for SNPs associated with spike-related traits. For instance, SNPs within csACRs of the WFZP and DUO1 promoters affect TaNAC30 binding, regulating supernumerary spikelet phenotypes. Our work provides a mechanistic framework for wheat inflorescence development and identifies csACRs and cTRN nodes as potential targets for optimizing yield-related inflorescence architecture.

Recent breakthroughs in spatial transcriptomics technologies have enhanced our understanding of diverse cellular identities, compositions, interactions, spatial organizations, and functions. Yet existing spatial transcriptomics tools are still limited in either transcriptomic coverage or spatial resolution. Leading spatial-capture or spatial-tagging transcriptomics techniques that rely on in-vitro sequencing offer whole-transcriptome coverage, in principle, but at the cost of lower spatial resolution compared to image-based techniques. In contrast, high-performance image-based spatial transcriptomics techniques, which rely on in situ hybridization or in situ sequencing, achieve single-molecule spatial resolution and retain sub-cellular morphologies, but are limited by probe libraries that target only a subset of the transcriptome, typically covering several hundred to a few thousand transcript species. Together, these limitations hinder unbiased, hypothesis-free transcriptomic analyses at high spatial resolution. Here we develop a new image-based spatial transcriptomics technology termed Reverse-padlock Amplicon Encoding FISH (RAEFISH) with whole-genome level coverage while retaining single-molecule spatial resolution in intact tissues. We demonstrate image-based spatial transcriptomics targeting 23,000 human transcript species or 22,000 mouse transcript species, including nearly the entire protein-coding transcriptome and several thousand long-noncoding RNAs, in single cells in cultures and in tissue sections. Our analyses reveal differential subcellular localizations of diverse transcripts, cell-type-specific and cell-type-invariant tissue zonation dependent transcriptome, and gene expression programs underlying preferential cell-cell interactions. Finally, we further develop our technology for direct spatial readout of gRNAs in an image-based high-content CRISPR screen. Overall, these developments provide the research community with a broadly applicable technology that enables high-coverage, high-resolution spatial profiling of both long and short, native and engineered RNA species in many biomedical contexts.

ABSTRACT Arterial diseases affect the mechanical properties of blood vessels, which then alter their function via complex mechanisms. To develop and test effective treatments, microphysiological systems replicating the function and mechanics of a human artery are needed. Here, we establish an artery-on-chip (ARTOC) using vascular derivatives of human induced pluripotent stem cells (iPSCs) cultured with pulsatile flow on an electrospun fibrin hydrogel. ARTOCs have mature, laminated smooth muscle that expresses robust extracellular matrix and contractile proteins, contracts in response to intraluminal pressure and vasoagonists, and exhibits tissue mechanics comparable to those of human small-diameter arteries. Using real-time monitoring of radial distention and luminal pressure to inform computational fluid dynamics modeling, we show that we can effectively tune biomechanical cues using fibrin scaffold thickness and luminal flow rate. We successfully tune these cues to promote the survival and function of both endothelial and smooth muscle cells simultaneously in the ARTOC. To test the ARTOC as a disease modeling platform, we first use non-isogenic iPSC-derived smooth muscle cells from a polycythemia patient, and we find significantly altered cell phenotype and increased vessel wall stiffness compared to controls. We then test a novel isogenic disease model in ARTOCs from iPSCs CRISPR-edited with the LMNA Hutchinson-Guilford Progeria Syndrome (LMNA G608G; LMNA HGPS ) mutation. LMNA HGPS ARTOCs show extracellular matrix accumulation, medial layer loss, premature senescence, and loss of tissue elasticity and ductility. With this work, we establish the ARTOC as a platform for basic and translational studies of arterial diseases, bridging the current gap in linking protein expression and cell phenotype to tissue mechanics and function in small-diameter arteries.

ABSTRACT The FOXG1 transcription factor is a crucial regulator of embryonic brain development. Pathogenic FOXG1 variants cause FOXG1 syndrome. Although structural variants (SVs) in the non-coding region downstream of FOXG1 have been reported in 38 individuals with similar characteristics, the regulatory pathomechanisms remain unknown. We identified a de novo non-coding deletion in an individual with FOXG1 syndrome-like disorder, allowing us to delineate a ∼124 kb commonly affected regulatory region (CARR). By integrating epigenomics data, 3D chromatin interaction profiles (Hi-C, UMI-4C), and in vivo enhancer assays in zebrafish, we uncovered multiple regulatory elements within this CARR, including a neuronal enhancer cluster and a conserved boundary of the FOXG1 -containing topologically associating domain (TAD). Hi-C analysis on case lymphoblastoid cells revealed increased interactions with the adjacent TAD. Moreover, sequential UMI-4C and CUT&RUN assays during neural progenitor cell (NPC) differentiation demonstrated dynamic activation of, and interaction with the enhancer cluster. Finally, CRISPR-Cas9 deletion of the enhancer cluster and TAD boundary in NPCs resulted in decreased FOXG1 transcription. We identified and characterized enhancer and architectural elements essential for proper FOXG1 transcription. Our findings provide new insights into chromatin architecture and gene regulation at the FOXG1 locus, improving SV interpretation in individuals with FOXG1 syndrome-like disorder.

Abstract In clinical settings, patients with SNTA1 point mutations are often associated with rare arrhythmias, including Long QT syndrome, Brugada syndrome, and sudden infant death syndrome. Previous studies on SNTA1 have predominantly utilized nonhuman cardiomyocyte models. This study aims to elucidate the phenotype of SNTA1 deficiency using human cardiomyocytes. Using CRISPR/Cas9 technology, we generated SNTA1 knockout (KO) embryonic stem cell line, which were subsequently differentiated into cardiomyocytes using 2D differentiation method. Genotype analysis identified an adenine (A) insertion in the second exon of SNTA1 , resulting in a premature stop codon at the 149th amino acid position and truncation within the PDZ domain. SNTA1 -deficient cardiomyocytes exhibited a shortened action potential duration (FPD) and slower conduction velocity, as detected by micro electrode array analysis. Immunofluorescence analysis further revealed disorganized distribution of SCN5A protein in SNTA1 -deficient cardiomyocytes. SNTA1 is a susceptibility locus for arrhythmias and plays a critical role as an essential auxiliary protein in the proper localization of SCN5A in human cardiomyocytes.

Genome-wide association studies (GWAS) have identified over a hundred genetic risk factors for Alzheimer’s disease (AD), many of which are predominantly expressed in microglia. However, the pathogenic role for most of them remains unclear. To systematically investigate how AD GWAS variants influence human microglial inflammatory responses, we conducted CRISPR inhibition (CRISPRi) screens targeting 119 AD GWAS hits in hiPSC-derived microglia (iMGLs) and used the production of reactive oxygen species (ROS) in response to the viral mimic poly(I:C) as a functional readout. Top hits whose knockdown either increased or decreased ROS levels in response to poly(I:C) were further analyzed using CROP-seq to integrate CRISPRi with single-cell RNA sequencing (scRNA-seq). This analysis identified 9 unique microglial clusters, including a poly(I:C)-driven inflammatory cluster 2. Emerging evidence supports a pathogenic role of viral infections in AD and cross comparison of our scRNA-seq data with iMGLs xenotransplanted into an AD mouse model shows significant overlap between our clusters and AD-relevant microglial clusters. Knockdown of MS4A6A and EED , which resulted in elevated ROS production in the presence of poly(I:C), increased the proportion of cluster 2 cells and induced functionally related changes in gene expression. In addition, KD of MS4A6 led to a reduction in the proportion of iMGLs in the DAM (disease associated microglia) cluster under all conditions, suggesting that this gene may modulate the DAM response. In contrast, KD of INPP5D or RAPEP1 which lead to low levels of ROS in the presence of poly(I:C), did not significantly affect the proportion of cells in cluster 2 but rather shaped the inflammatory response. This included the upregulation of an HLA-associated inflammatory cluster (cluster 6) by INPP5D knockdown under all conditions, independent of poly(I:C) stimulation. Importantly, KD of INPP5D or RAPEP1 had many shared differentially expressed genes (DEGs) under both vehicle and poly(I:C) treated conditions. Overall, our findings demonstrate that despite the diverse biological functions of AD GWAS variants, they converge functionally to regulate human microglial states and shape inflammatory responses relevant to AD pathology.

No method has been developed for single-cell analysis of the large repositories of preserved whole blood samples stored in PAXgene Blood RNA tubes. To address this gap, two nuclei isolation techniques for single-nucleus RNA sequencing were evaluated: mechanical separation (MS), using an Acrodisc filter, and cell lysis (CL). While both methods captured nuclei from all major immune cell types, CL resulted in up to two orders of magnitude higher nuclei yields and less biased proportions of immune cells than MS. High ambient globin gene counts following CL were substantially reduced by CRISPR-guided globin gene depletion of complementary DNA, resulting in more sensitive and efficient gene detection per cell. Despite capturing only nuclear transcripts, CL-derived samples maintained similar cell-type proportions and gene expression as matched peripheral blood mononuclear cell samples, while retaining granulocytes. The CL isolation with globin depletion method enables comprehensive analysis of PAXgene whole blood samples at single-cell resolution. MOTIVATION PAXgene Blood RNA Tubes are commonly used for blood preservation and gene expression studies due to the ease of use and stabilization of RNA. To date, transcriptomic studies using PAXgene tubes have been limited to bulk RNA profiling approaches such as RNA sequencing (RNA-seq). Here we introduce a method for recovering high-quality nuclei from frozen blood preserved in PAXgene tubes for single-nucleus RNA-seq (snRNA-seq). Using this approach, all white blood cell types – including granulocytes – are captured, thereby enabling comprehensive transcriptomic profiling of peripheral blood at single-cell resolution.

ABSTRACT N-glycanase 1 (NGLY1) deficiency is an ultra-rare disease caused by autosomal recessive loss-of-function mutations in the NGLY1 gene. NGLY1 removes N-linked glycans from glycoproteins in the cytoplasm and is thought to help clear misfolded proteins from the endoplasmic reticulum (ER) through the ER associated degradation (ERAD) pathway. Despite this, the physiological significance of NGLY1 in ERAD is not understood. The best characterized substrate of NGLY1 is NRF1, a transcription factor that upregulates proteasome expression and the proteasome bounce-back response. We previously performed a genetic modifier screen using a Drosophila model of NGLY1 deficiency and identified potential modifiers that alter the lethality of the model. We identified two protein-coding variants in Hrd3 / SEL1L S780P and Δ806-809 . Both variants are localized to the SEL1L cytoplasmic tail, an uncharacterized domain. SEL1L is a component of the ERAD complex that retrotranslocates misfolded proteins from the ER to the cytoplasm for degradation. We used CRISPR to generate fly lines carrying these SEL1L variants in a common genetic background and tested them with our model of NGLY1 deficiency. Validating our previous screen, the SEL1L P780 and SEL1L Δ806-809 variants increase the survival of the NGLY1 deficiency model, compared to the SEL1L S780 variant. To determine how these SEL1L variants were modifying lethality in NGLY1 deficiency, we interrogated the ERAD and NRF1 signaling pathways. We found that the SEL1L P780 and SEL1L Δ806-809 variants improve ERAD function in an NGLY1- dependent manner, further implicating NGLY1 in general ERAD function. We also found that these variants protect against changes in larval size and survival caused by proteasome inhibition in heterozygous NGLY1 null flies. These results provide new insights into the role of SEL1L in the disease pathogenesis of NGLY1 deficiency. SEL1L is a strong candidate modifier gene in patients, where variability in presentation is common. AUTHOR’S SUMMARY NGLY1 deficiency is a debilitating rare genetic disorder. There are currently no treatment options for NGLY1 deficiency and NGLY1 biology remains poorly understood. We previously performed a genetic modifier screen in a Drosophila model of NGLY1 deficiency and identified a number of candidate modifier genes that impacted the survival of our model. Modifier genes can help reveal NGLY1 biology and NGLY1 deficiency disease pathogenesis. In this study, we follow-up on two natural protein-coding variants of Hrd3 (the fly version of the human gene, SEL1L ) that increased the survival of our NGLY1 deficiency model. SEL1L is a critical component of an important quality control pathway called the endoplasmic reticulum associated degradation (ERAD) pathway. We discovered that these SEL1L variants enhance ERAD and modify NGLY1 deficiency sensitivity to proteasome inhibition. This study confirms SEL1L as an important modifier gene of NGLY1 deficiency. Further study of the ERAD and proteasome degradation pathways may reveal additional candidate modifier genes of NGLY1 deficiency and potential targets for therapeutic development.

ABSTRACT Cap-adjacent 2’- O -ribose methylation (cOMe) of the first two transcribed nucleotides of RNA polymerase II transcripts is a conserved feature in many eukaryotes. In mammals, these modifications are key to a transcript surveillance system that regulates the interferon response, but the broader functions of cOMe remain poorly understood. To understand the role of cOMe in C. elegans , we functionally characterised the methyltransferases (CMTR-1 and CMTR-2) responsible for installing these modifications. These enzymes have distinct expression patterns, protein interaction partners, and loss of function phenotypes. Loss of CMTR-1 causes dramatic reductions in cOMe, impaired growth and sterility. In contrast, animals lacking CMTR-2 are superficially wild-type, though CMTR-2 loss enhances the severity of the cmtr-1 mutant phenotype. Depletion of CMTR-1 causes downregulation of transcripts associated with germline sex determination and upregulation of those involved in the intracellular pathogen response (IPR). We show that absence of the decapping exonuclease, EOL-1, an IPR component, completely suppresses the sterility and growth defects caused of loss of CMTR-1, suggesting that EOL-1 degrades cellular transcripts lacking cOMe. Our work shows the physiological relevance of cOMe in protecting transcripts from decapping exonucleases, raising the possibility that cOMe plays a role in RNA-mediated immune surveillance beyond the vertebrates. GRAPHICAL ABSTRACT

Comprehensive and systematic proteome-wide experiments require financial and technical resources unavailable to most researchers. Here we describe a scalable CRISPR/Cas9-non-homologous end joining (NHEJ) based method for P ooled R ecombinant I ntegration of S eamless M arkers (PRISM) into protein coding genes. We created two gRNA libraries for 5’- and 3’-tagging of 18,804 human protein-coding genes. Selection for in-frame integration of the donor cassette can be guaranteed by fusing it to an antibiotic resistance enzyme (ARE) and P2A self-cleaving peptide, resulting in tagged proteins and free ARE to select for clones with inserted donor cassettes. We achieved a library integration rate of 19.75% and tagging of ∼80% for genes expressed in Hek293T cells and notably, 89.7% of essential genes, with donor DNA. Our strategy is scalable, specific, and selective, paving the way for genome-scale construction of human cell lines tagged with different types of reporter genes for protein functional characterization. Significance statement Here we report the first practical method to achieve near complete 5’- or 3’-end integration of reporter protein-coding sequences to express seamless fusions of human protein and reporter proteins that we call P ooled R ecombinant I ntegration of S eamless M arkers ( PRISM ). PRISM is independent of homology templates, yet it works with high efficiency and precision. Using gRNA-directed CRISPR/Cas9 genome editing and mini-plasmid donor cassettes we were able to tag about 80% of the protein-coding sequences of human genes with a green florescent protein at an integration efficiency of about 20% and most notably, of 90% of essential genes in the human cell line HEK 293T. This is important because the characterization of gene functions in a particular cell type usually begins with essential genes.

ABSTRACT Enterococcus faecalis is a Gram-positive bacterium and opportunistic pathogen that acquires resistance to a wide range of antibiotics by horizontal gene transfer (HGT). The rapid increase of multidrug-resistant (MDR) bacteria including MDR E. faecalis necessitates the development of alternative therapies and a deeper understanding of the factors that impact HGT. CRISPR-Cas systems provide sequence-specific defense against HGT. From previous studies, we know that E. faecalis CRISPR-Cas provides sequence-specific anti-plasmid defense during agar plate biofilm mating and in the murine intestine. Those studies were mainly conducted using laboratory model strains with a single, CRISPR-targeted plasmid in the donor. MDR E. faecalis typically possess multiple plasmids that are diverse in sequence and may interact with each other to impact plasmid transfer and CRISPR-Cas efficacy. Here, we altered multiple parameters of our standard in vitro conjugation assays to assess CRISPR-Cas efficacy, including the number and genotype of plasmids in the donor; and laboratory model strains as donor versus recent human isolates as donor during conjugation. We found that the plasmids pTEF2 and pCF10, which are not targeted by CRISPR-Cas in our recipient, enhance the conjugative transfer of the CRISPR-targeted plasmid pTEF1 into both wild-type and CRISPR-Cas-deficient (via deletion of cas9 ) recipient cells. However, the effect of pTEF2 on pTEF1 transfer is much more pronounced, with a striking 6-log increase in pTEF1 conjugation frequency when pTEF2 is also present in the donor and recipients are deficient for CRISPR-Cas (compared to 4-log for pCF10). Overall, this study provides insight about the interplay between plasmids and CRISPR-Cas defense, opening avenues for developing novel therapeutic strategies to curb HGT among bacterial pathogens, and highlighting pTEF2 as a plasmid for additional mechanistic study. IMPORTANCE The emergence of MDR bacteria, including MDR E. faecalis, limits treatment options and necessitates development of alternative therapeutics. In these circumstances, bacterial CRISPR-Cas systems are being explored by the field to develop CRISPR-based antimicrobials. However, in many cases CRISPR-Cas efficacy has only been assessed using laboratory model strains. More studies are required that investigate clinical isolates, as those are the intended targets for CRISPR antimicrobials. Here, we demonstrated how the number of plasmids harbored by an E. faecalis donor strain can affect the apparent efficacy of CRISPR-Cas anti-plasmid defense in a recipient strain. Overall, our research is important to develop improved CRISPR-based antimicrobials to combat the spread and accumulation of antibiotic resistance determinants.

RNA therapeutics are emerging as transformative modalities in clinical applications and have become a key area in life science research. While lipid nanoparticles (LNPs) have become the leading platform for RNA delivery in preventive and therapeutic products, they still face significant challenges in achieving efficient extrahepatic delivery and maintaining long term shelf stability. Here we report the development of a series of biodegradable poly( β -amino amide) (PBAA) polymers, detailing their design, synthesis, and performance as gene delivery vehicles both in vitro and in vivo . These cationic polymers, featuring interspersed trialkylamine motifs, provide a readily tunable functional handle and facilitate complexation with gene cargo. The redox-sensitive disulfide motifs introduce a redox-responsive decomposition pathway for the polymer backbone, triggering cargo release during intracellular delivery. The results herein demonstrate that these polymers offer remarkable efficiency in encapsulating and delivering translation-competent cargos, including mRNA and CRISPR-Cas based gene editing tools. Notably, a dodecyl modified PBAA transporter has achieved over 97% gene editing efficiency in vitro , and over 97% spleen-targeting selectivity in a murine model. Additionally, it produces stable nanoparticles that maintain their physicochemical properties at 4 °C for up to two weeks without addition of excipients such as PEG, offering a cost-effective solution for RNA therapeutics supply chains. As the transformative impact of RNA and other nucleic acids continues to build within the pharmaceutical industry and beyond, it is imperative that the supporting technologies evolve in stride to maximize said impact. The tunable and biodegradable PBAA polymer designs presented herein are illustrative examples of how high-level functional performance can be acheived in conjunction with the critical targeting, formulation, and operational simplicity needed of state-of-the-art transfection and delivery technologies.

Bacillus thuringiensis is widely employed for biological control. It can effectively suppress populations of various mosquito species, including Aedes aegypti . However, the precise mechanism underlying the action of cry toxin secreted by Bacillus thuringiensis on Ae. aegypti remains elusive. In this study, we investigated one of the binding receptors of cry toxin, aminopeptidase N. Through comprehensive bioinformatics analysis involving whole-genome screening, genetic mapping, structural characterization, phylogenetic analysis, and spatiotemporal expression profiling, we identified twenty-nine homologs of Ae. aegypti aminopeptidase N. Further, we successfully expressed GST-APN3 protein in E. coli and demonstrated through ligand blot and ELISA assays that APN3 exhibits high affinity binding to Cry4Ba toxin (Kd = 20.53 nM). To elucidate the functional role of APN3 as a receptor mediating Cry4Ba activity in Ae. aegypti midgut cells (some of which express this gene at high levels), CRISPR/Cas9 technology was employed to knock out APN3. Our bioassay results revealed that APN3 knockout mosquito larvae had 2.9 to 4.1-fold higher resistance against Cry4Ba, indicating its crucial involvement as an active receptor mediating Cry4Ba activity. Overall, this study provides a foundation for elucidating the specific larvicidal mechanisms of Bt against mosquito populations.

Background Endozoicomonas is a widely distributed genus of marine bacteria, associated with various marine organisms, and recognized for its ecological importance in host health, nutrient cycling, and disease dynamics. Despite its significance, genomic features of Endozoicomonas remain poorly characterized due to limited availability of high-quality genome assemblies. Results In this study, we sequenced 5 novel Endozoicomonas strains and re-sequenced 1 known strain to improve genomic resolution. By integrating these 6 high-quality genomes with 31 others that were publicly available, we identified a distinct, coral-associated clade not recognized by the previous two-clade classification. Pan-genomic analysis revealed significant variation in genetic trait distribution among clades. Notably, Endozoicomonas lacks quorum sensing capabilities, suggesting resistance to quorum quenching mechanisms. It also lacks the ability to synthesize and transport vitamin B12, indicating that it is not a primary source of this nutrient for holobionts. A remarkable feature of Endozoicomonas is its abundance of giant proteins, ranging from 15 to 65 kbp. We identified 92 such proteins, which clustered into three major groups based on amino acid similarity, each associated with specialized functions, such as antimicrobial synthesis, exotoxin production, and cell adhesion. Additionally, we explored prophages and CRISPR-Cas systems. We found that Endozoicomonas acquired prophages from diverse sources via infection or other types of gene transfer. Notably, CRISPR-Cas sequences suggest independent evolutionary trajectories from both prophage acquisition and phylogenetic lineage, implying a potential influence of geographic or environmental pressures. Conclusions This study provides new insights into the genomic diversity of Endozoicomonas and its genetic adaptation to diverse hosts. Identification of novel genomic features, including deficiencies in B12 synthesis and quorum sensing, the presence of giant proteins, prophages, and CRISPR-Cas systems, underscores its ecological roles in various holobionts. These findings open new avenues for research on Endozoicomonas and its ecological interactions.

Heart failure (HF) is a global health challenge characterized by the heart’s inability to satisfy metabolic demands, driven by renin-angiotensin-aldosterone system (RAAS) overactivation, neurohormonal imbalance, and emerging mechanisms like the gut-heart axis and mitochondrial dysfunction. Affecting over 6 million adults in the US alone, HF incurs a 5-year mortality rate of 50% and escalating costs projected to double by 2030. This review examines HF’s molecular paradigms, integrating established pathways with advances in omics, stem cell therapy, genetic modification, and personalized medicine. RAAS blockade remains central, yet its efficacy is limited in HF with preserved ejection fraction (HFpEF). Stem cell therapies (mesenchymal and induced pluripotent stem cells) show regenerative potential but face poor retention (10% survival at 30 days). CRISPR/Cas9 offers precision, though off-target effects persist. The gut microbiome, via trimethylamine N-oxide, exacerbates inflammation, while omics technologies promise biomarkers for tailored treatments. Challenges include translating these innovations into practice, particularly for HFpEF. Future directions involve novel HFpEF therapies, enhanced stem cell delivery, precise genetic tools, and microbiome interventions, supported by artificial intelligence. By 2030, these advances could shift HF management toward regeneration, contingent on overcoming translational barriers through global collaboration.

Mycobacterium abscessus (Mab) causes pulmonary diseases with limited treatment options due to its high level of intrinsic resistance to available drugs. Mab possesses complex and poorly understood drug resistance mechanisms. Identifying new drug targets and gaining a deeper understanding of drug resistance mechanisms are essential for discovering novel therapeutic alternatives. Here, we investigated the role of a putative sigma factor SigH in intrinsic multi-drug resistance in Mab. Mab SigH shares an 84% peptide sequence identity with Mycobacterium tuberculosis (Mtb) SigH, a well-known stress response protein and global transcriptional regulator. We constructed a sigH gene deletion strain of Mab (Δ sigH ) and complemented strains by expressing either Mab sigH (CPMab sigH ) or Mtb sigH (CPMtb sigH ) in Δ sigH. The Δ sigH strain exhibited hypersensitivity to a broad range of antibiotics, including levofloxacin, moxifloxacin, tigecycline, tetracycline, amikacin, vancomycin, and rifabutin and all complemented strains restored the drug resistance phenotype. Additionally, Δ sigH showed increased sensitivity to oxidative and heat stress compared to the wild-type Mab and complemented strains. Transcriptomic analysis revealed that deletion of sigH disrupted the balance of gene expression, primarily elevating the expression of genes encoding YrbE and MCE family proteins and downregulating genes expressing ABC-type transporters, sigma and anti-sigma factors and other genes associated with antimicrobial resistance. Collectively, our findings indicate that SigH is a key regulator of global gene expression in response to environmental stresses, including antimicrobial treatment, and is crucial for the intrinsic drug resistance of Mab. SigH represents a promising target for the development of novel therapeutic strategies against Mab infections.

Lipid nanoparticle (LNP)-based mRNA therapeutics, highlighted by the success of SARS-CoV-2 vaccines, face challenges due to inflammation caused by ionizable lipids. These ionizable lipids can activate the immune system, particularly when co-delivered with nucleic acids, leading to undesirable inflammatory responses. We introduce a novel class of anti-inflammatory ionizable lipids functionalized with hydroxychloroquine (HCQ), which suppresses both lipid-induced and nucleic acid-induced immune activation. These HCQ-functionalized LNPs (HL LNPs) exhibit reduced proinflammatory responses while maintaining efficient mRNA delivery. Structural and physicochemical analyses revealed that HCQ-functionalization results in a distinct particle structure with significantly improved stability. The efficacy of HL LNPs was demonstrated across various therapeutic contexts, including a prophylactic vaccination model against varicella-zoster virus (VZV) and CRISPR-Cas9 gene editing targeting PCSK9. Notably, HL LNPs showed robust mRNA expression after repeated administration, addressing concerns of inflammation and ensuring sustained therapeutic effects. These findings highlight the potential of HCQ-functionalized LNPs in expanding the safe use of mRNA therapeutics, particularly for applications requiring repeated dosing and in scenarios where inflammation-induced side effects must be minimized.

Streptomyces bacteria make diverse specialised metabolites that form the basis of ∼55% of clinically used antibiotics. Despite this, only 3% of their encoded specialised metabolites have been matched to molecules and understanding how their biosynthesis is controlled is essential to fully exploit their potential. Here we use Streptomyces formicae and the formicamycin biosynthetic pathway as a model to understand the complex regulation of specialised metabolism. We analysed all three pathway-specific regulators and found that biosynthesis is subject to negative feedback and redox control via two MarR-family proteins while activation of the pathway is dependent on a cytoplasmic two-component system. Like many Streptomyces antibiotics, formicamycins are only produced in solid culture and biosynthesis is switched off in aerated liquid cultures. Here, we demonstrate that a redox-sensitive repressor named ForJ senses oxygen via a single cysteine residue that is required to repress formicamycin biosynthesis in liquid cultures.

CRISPR-associated transposons (CAST) consist of an integration between certain class 1 or class 2 CRISPR-Cas systems and Tn7-like transposons. Class 2 type V-K CAST systems are restricted to cyanobacteria. Here, we identified a unique subgroup of type V-K systems through phylogenetic analysis, classified as V-K_V2. Subgroup V-K_V2 CAST systems are characterized by an alternative tracrRNA, the exclusive use of Arc_2-type transcriptional regulators, and distinct differences in TnsB and TnsC proteins. Although the occurrence of V-K_V2 CAST systems is restricted to Nostocales cyanobacteria, it shows signs of horizontal gene transfer, indicating its capability for genetic mobility. The predicted V-K_V2 tracrRNA secondary structure has been integrated into an updated version of the CRISPRtracrRNA program available on GitHub under https://github.com/BackofenLab/CRISPRtracrRNA/releases/tag/2.0 .

Abstract The accumulation of misfolded protein species underlies a broad range of neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS). Due to their dynamic nature, these misfolded proteins have proven challenging to target therapeutically. Here, we specifically target misfolded disease variants of the ALS-associated protein; SOD1, using a biological proteolysis targeting chimera (BioPROTAC) composed of a SOD1-specific intrabody and an E3 ubiquitin ligase. Screening of intrabodies and E3 ligases for optimal BioPROTAC construction revealed a candidate capable of degrading multiple disease variants of SOD1, preventing their aggregation in cultured cells. Using CRISPR/Cas9 technology to develop a BioPROTAC transgenic mouse line, we demonstrate that the presence of the BioPROTAC delays disease progression in the SOD1G93A mouse model of ALS. Delayed disease progression was associated with protection of motor neurons, reduced accumulation of insoluble SOD1 and protection of neuromuscular junctions. These findings provide proof-of-concept evidence and a platform for developing BioPROTACs as a therapeutic strategy for the targeted degradation of neurotoxic misfolded species in the context of neurodegenerative diseases.

Hypophosphatasia (HPP) is a rare inborn error of metabolism caused by pathogenic var-iants in ALPL, coding for tissue non-specific alkaline phosphatase. HPP patients suffer from impaired bone mineralization and in severe cases from vitamin B6-responsive sei-zures. To study HPP we generated alpl-/- zebrafish line using CRISPR/Cas9 gene-editing technology. At 5 days post fertilization (dpf) no alpl mRNA and 89% lower total alkaline phosphatase activity was detected in alpl-/- compared to alpl+/+ embryos. The survival of alpl-/- zebrafish was strongly decreased. Alizarin red staining showed decreased bone mineralization in alpl-/- embryos. B6 vitamer analysis revealed depletion of pyridoxal and its degradation product 4-pyridoxic acid in alpl-/- embryos. Accumulation of d3-pyridoxal 5’-phosphate (d3-PLP) and reduced formation of d3-pyridoxal in alpl-/- embryos incu-bated with d3-PLP confirmed Alpl involvement in vitamin B6 metabolism. Locomotion analysis showed pyridoxine treatment-responsive spontaneous seizures in alpl-/- embryos. Metabolic profiling of alpl-/- larvae using direct-infusion high-resolution mass spectrome-try showed abnormalities in polyamine and neurotransmitter metabolism, suggesting dysfunction of vitamin B6-dependent enzymes. Accumulation of N-methylethanolaminium phosphate indicated abnormalities in phosphoethanolamine metabolism. Taken together, we generated first zebrafish model of HPP that shows multi-ple features of human disease and is suitable to study pathophysiology of HPP and to test novel treatments.

Intratumor transcriptional heterogeneity (ITTH) presents a major challenge in cancer treatment, particularly due to limited understanding of the diverse malignant cell populations and their relationship to therapy resistance. While single-cell sequencing has provided valuable insights into tumor composition, its high cost and technical complexity limits its use for large-scale tumor screening. In contrast, several databases collecting bulk RNA sequencing (RNA-seq) data from multiple samples across various cancer types are available and could be used to profile ITTH. Several deconvolution approaches have been developed to infer cellular composition from such data. However, most of these methods rely on predefined markers or single cell reference datasets, limiting the performance of such methods by the quality of used reference data. Although unsupervised approaches do not face such limitations, existing methods have not been specifically adapted to characterize malignant cell states, and focus instead on general cell types. To address these gaps, we introduce CDState, an unsupervised method for inferring malignant cell subpopulations from bulk RNA-seq data. CDState utilizes a Nonnegative Matrix Factorization model improved with sum-to-one constraints and a cosine similarity-based optimization to deconvolve bulk gene expression into distinct cell state-specific profiles, and estimate the abundance of each state across tumor samples. We validate CDState using bulkified single-cell RNA-seq data from five cancer types, showing that it outperforms existing unsupervised deconvolution methods in both cell state proportions and gene expression estimation. Applying CDState to 33 cancer types from TCGA, we identified recurrent malignant cell programs, with epithelial-mesenchymal transition as the main driver of tumor transcriptional heterogeneity. We further link the identified malignant states to patient clinical features, revealing states associated with worse patient prognosis. We show that ITTH is linked with patient survival and clinical features, as well as is associated with varied responses to therapy. Finally, we identified potential genetic drivers of ITTH and malignant cell states, including TP53 , KRAS , and PIK3CA , known oncogenic genes.

Cis-regulatory elements (CREs) control how genes respond to external signals, but the principles governing their structure and function remain poorly understood. While differential transcription factor binding is known to regulate gene expression, how CREs integrate the amount and combination of inputs to secure precise spatiotemporal profiles of gene expression remains unclear. Here, we developed a high-throughput combinatorial screening strategy, that we term NeMECiS, to investigate signal- dependent synthetic CREs (synCREs) in differentiating mammalian stem cells. By concatenating fragments of functional CREs from genes that respond to Sonic Hedgehog in the developing vertebrate neural tube, we found that CRE activity follows hierarchical design rules. While individual 200-base-pair fragments showed minimal activity, their combinations generated thousands of functional signal-responsive synCREs, many exceeding the activity of natural sequences. Statistical modelling revealed CRE function can be decomposed into specific quantitative contributions in which sequence fragments combine through a multiplicative rule, tuned by their relative positioning and spacing. These findings provide a predictive framework for CRE redesign, which we used to engineer synthetic CREs that alter the pattern of motor neuron differentiation in neural tissue. These findings establish quantitative principles for engineering synthetic regulatory elements with programmable signal responses to rewire genetic circuits and control stem cell differentiation, providing a basis for understanding developmental gene regulation and designing therapeutic gene expression systems.

We established a novel knock-in technique, New and Easy Xenopus Targeted integration ( NEXTi ), to recapitulate endogenous gene expression by reporter expression. NEXTi is a CRISPR-Cas9-based method to integrate a donor DNA containing a reporter gene ( egfp ) into target 5’ untranslated region (UTR) of Xenopus laevis genome. It enables us to track eGFP expression under regulation of endogenous promoter/enhancer activities. We obtained about 2% to 13% of knock-in vector-injected embryos showing eGFP signal in a tissue-specific manner, targeting krt.12.2.L , myod1.S , sox2.L and bcan.S loci, as previously reported. In addition, F1 embryos which show stable eGFP signals were obtained by outcrossing the matured injected frogs with wild-type animals. Integrations of donor DNAs into target 5’ UTRs were confirmed by PCR amplification and sequencing. Here, we describe the step-by-step protocol for preparation of donor DNA and single guide RNA, microinjection and genotyping of F1 animals for the NEXTi procedure. 1. Highlight Intended organism: Xenopus laevis Purpose: of the protocol: Efficient knock-in for visualizing endogenous target gene expression using CRISPR-Cas9 system Essential equipment and materials: Microinjector, fluorescence microscopy, Cas9 protein, sgRNA, donor DNA Features: Expression of reporter genes depends on endogenous enhancer/promoter activities.

Hypoxia, a hallmark of solid tumors, promotes the malignant progression and is challenging to target. Metabolic reprogramming and the resulting metabolic vulnerabilities provide a promising strategy to target tumor hypoxia. Here we systematically compared the metabolic network differences between hypoxic and non-hypoxic cells, and developed a deep learning model, “DepFormer”, to predict the dependent metabolic genes in hypoxic tumor cells. The performance of DepFormer was validated using CRISPR screening dataset. Oxidative phosphorylation was identified as the most significantly hypoxia-dependent metabolic pathway, and FLAD1 was predicted to be one of the key hypoxia-dependent metabolic genes. FLAD1 locus is amplified, and FLAD1 expression is upregulated in various tumor types, especially in hypoxic tumors. FLAD1 depletion compromises tumor’s adaptation to hypoxia by disrupting mitochondrial complex II activity, leading to an imbalance between succinate and fumarate, and consequent failure to adapt to hypoxia. Subsequently, we identified a drug-like inhibitor of FLAD1, which selectively inhibits the growth of tumor cells under hypoxia. Our findings reveal FLAD1 as an innovative therapeutic target for hypoxic tumors.