Nucleotide-binding domain and leucine-rich repeat immune receptors (NLRs) can function in networks of sensors and helpers to induce hypersensitive cell death and immunity against pathogens. The tomato sensor NLR Prf guards the Pto kinase from AvrPto and AvrPtoB effector perturbation and activates the downstream helpers NRC2 and NRC3. Prf is conserved across the Solanaceae and its ortholog in the model species Nicotiana benthamiana is also required for detection of AvrPto/AvrPtoB function on Pto. A recent study reported that cell death induction after transient expression of an autoactive mutant of tomato NRC3 is abolished upon RNAi silencing of Prf in N. benthamiana . Here we generated loss-of-function prf mutants in N. benthamiana and demonstrate that autoactive mutants of eight canonical tomato NRCs (NRC0, NRC1, NRC2, NRC3, NRC4a, NRC4b, NRC6, and NRC7) still induce hypersensitive cell death when expressed transiently in the prf mutant background. Autoactive tomato NRCs also triggered cell death when expressed in lettuce ( Lactuca sativa ), an Asteraceae plant that does not have a Prf ortholog. These results confirm a unidirectional dependency of sensors and helpers in the NRC network and underscore the value of the N. benthamiana and lettuce model systems for studying functional relationships between paired and networked NLRs.

ABSTRACT Inhibitors of the protein kinase WEE1 have emerged as promising agents for cancer therapy. In this study, we uncover synergistic interactions between WEE1 small- molecule inhibitors and defects in mRNA translation, mediated by activation of the integrated stress response (ISR) through the kinase GCN2. Using a pooled CRISPRi screen, we identify GSPT1 and ALKBH8 as factors whose depletion confer hypersensitivity to the WEE1 inhibitor, AZD1775. We demonstrate that this synergy depends on ISR activation, which is induced by the off-target activity of WEE1 inhibitors. Furthermore, PROTAC-based WEE1 inhibitors and molecular glues show reduced or no ISR activation, suggesting potential strategies to minimise off-target toxicity. Our findings reveal that certain WEE1 inhibitors elicit dual toxicity via ISR activation and genotoxic stress, with ISR activation being independent of WEE1 itself or cell-cycle status. This dual mechanism highlights opportunities for combination therapies, such as pairing WEE1 inhibitors with agents targeting the mRNA translation machinery. This study also underscores the need for more precise WEE1 targeting strategies to mitigate off-target effects, with implications for optimising the therapeutic potential of WEE1 inhibitors.

Cassava production in sub-Saharan Africa is severely impacted by diseases. Most pathogens require interaction with host susceptibility factors to complete their life cycles and cause disease. Targeted DNA methylation is an epigenetic strategy to alter gene expression in plants and we previously reported that a zinc-finger fused to DMS3 could establish methylation at the promoter of MeSWEET10a , a bacterial susceptibility gene, and this resulted in decreased disease. Here, we attempt a similar strategy for cassava brown streak disease. This disease is caused by the ipomoviruses CBSV and UCBSV. These viruses belong to the family Potyviridae , which has been shown extensively to require host eIF4E-family proteins to infect plants and cause disease. We previously found that cassava plants with simultaneous knockout mutations in two eIF4E genes, nCBP-1 and nCBP-2 , resulted in decreased susceptibility to CBSD. Here, we report successful simultaneous targeting of both promoters with methylation using a dCas9-DMRcd-SunTag system. However, in contrast to our previous work with MeSWEET10a , controls indicate that CRISPR interference is occurring in these lines and is sufficient for reduction of gene expression. Future research will use genetic crosses to segregate away the DNA methylation reagents and, if DNA methylation proves heritable, assess whether methylation alone is sufficient increase resistance to CBSD.

The size distribution of starch granules in wheat grains influences bread- and pasta-making quality, as well as nutritional properties. Here, we demonstrate that in durum wheat, wide variation in starch granule size distributions can be induced through missense mutations at a single genetic locus encoding the MYOSIN RESEMBLING CHLOROPLAST PROTEIN on chromosome 6A ( TtMRC-A1 ). We isolated 29 independent TILLING mutants in durum cultivar Kronos, each harbouring a different missense mutation that causes an amino acid substitution in the MRC protein. Compared to the B-type granule content of wild-type Kronos (24%), six of the missense lines had significant increases in B-type granule content (33-42%), although not to the extent observed in the mrc-1 mutant (58%) which carries a premature stop codon mutation. Notably, one missense line had significantly decreased B-type granule content (15%), demonstrating that mutations in TtMRC-A1 can achieve both increases and decreases in B-type granule content. In these lines, A-type granule size decreased as B-type granule content increased, and Rapid Visco Analysis on selected lines demonstrated that both B-type granule content and A-type granule size strongly correlated with pasting parameters (e.g., peak viscosity and pasting temperature). However, strong correlations between pasting properties and A-type granule size were still observed after removing most of the B-type granules via sieving, indicating that A-type granule size is the primary contributor to the observed variation in pasting properties. Overall, we demonstrate that mutations at TtMRC-A1 can greatly extend the range of granule size distributions in durum wheat, creating useful alterations in starch properties. Key Message Different missense mutations in TtMRC-A1 can be used to fine tune granule size distributions in durum wheat grains, creating useful alterations in starch properties.

Pennycress is a winter annual intermediate crop with approximately 30% seed oil content suitable for producing biofuels. Here, we evaluated seed development, anatomy, and agronomically relevant traits of a transparent testa 8 knockout mutant ( tt8-2bp ) generated by CRISPR genome editing to improve seed quality. We performed histochemical analyses on wild-type and tt8-2bp seeds at different developmental stages. No visible anatomical defects were observed in embryos and endosperm of tt8-2bp seeds. However, tt8-2bp seed coats completely lost proanthocyanidins which were accumulated in an inner integument cell layer and in the thickened cell wall of an outer integument cell layer of wild-type seed coats. Based on spatial metabolomic and solid-state NMR analyses, tt8-2bp seed coats had decreased aromatic compounds and cell wall polysaccharides compared to wild-type seed coats. Additionally, tt8-2bp seeds had reduced seed coat dry weights and increased embryo dry weights compared to wild-type seeds, indicating changes in macronutrient partitioning during seed development. Mature tt8-2bp seeds exhibited increased imbibition rates and seed coat permeability to water-soluble molecules, suggesting a higher seed coat hydrophilicity than wild-type seeds. In conclusion, we did not find defects in tt8-2bp mutant seeds that were unfavorable agronomically, supporting that TT8 is an attractive target for pennycress domestication. Highlight Histochemical analyses of pennycress seeds revealed a complete loss of proanthocyanidins in tt8-2bp seed coats accompanied by increased seed imbibition rates and seed coat permeability compared to wild-type seeds.

ABSTRACT The regulation of cell division is broadly conserved across eukaryotes, governed by cyclins and cyclin-dependent kinases (CDKs) to coordinate progression through the cell cycle. Plants have evolved a complex set of cell-cycle genes with unique features. The high number of cyclin-CDK pairs in flowering plants complicates functional studies due to redundancy and diversification. It is critical to study simple systems in other plant lineages to better understand the functional integration of the cell-cycle control machinery and its evolution across land plants. Through a comprehensive phylogenetic analysis, we show that non-seed plants possess a simple repertoire of cyclin and CDK proteins, suggesting that the observed complexity in seed plants is a derived trait. The liverwort Marchantia polymorpha possesses a streamlined set of core cell cycle genes with minimal redundancy during vegetative development. Using single-cell RNA-seq and fluorescent reporters, we found a precise, phase-specific expression pattern for cell cycle genes. We demonstrated in vivo that only three cyclins are active, one at a given phase, without redundancy. Functional analyses revealed that Mp CYCD;1 promotes cell cycle re-entry and disrupts differentiation, while overexpression of Mp CYCA or Mp CYCB;1 arrests the cell cycle, consistent with their respective roles at G1, S, and G2/M progression. Our findings highlight the functional conservation of mechanisms for cell-cycle control across eukaryotes and provide insights into its ancestral state, revealing a minimal set of functional components required for multicellular development. This study advances our understanding of fundamental aspects of cell-cycle regulation and opens new possibilities for engineering plant growth.

Microbial host populations evolve traits conferring specific resistance to viral predators via various defense mechanisms, while viruses reciprocally evolve traits to evade these defenses. Such co-evolutionary dynamics often involve diversification promoted by negative frequency-dependent selection. However, microbial traits conferring competitive asymmetries can induce directional selection, opposing diversification. Despite extensive research on microbe-virus co-evolution, the combined effect of both host trait types and associated selection remains unclear. Using a CRISPR-mediated co-evolutionary system, we examine how the co-occurrence of both trait types impacts viral evolution and persistence, previously shown to be transient and non-stationary in computational models. A stochastic model incorporating host competitive asymmetries via variation of intrinsic growth rates reveals that competitively-advantaged host clades generate the majority of immune diversity. Greater asymmetries extend viral extinction times, accelerate viral adaptation locally in time, and augment long-term local adaptation. These findings align with previous experiments, and provide further insights into long-term co-evolutionary dynamics.

ATRX is a member of the SWI/SNF family of ATP-dependent chromatin remodellers. In humans, loss of ATRX function leads to ATRX syndrome, a neurodevelopmental disorder. ATRX mutation in human cell lines is associated with multiple phenotypes including activation of the alternative lengthening of telomere (ALT) pathway, upregulation of retrotransposons and increased sensitivity to replication stress. However, the principal role of ATRX and the reason why its mutation causes such diverse phenotypes is currently unclear. To address this, we studied the role of ATRX in the model organism Caenorhabditis elegans . We find that loss of XNP-1, the C. elegans homologue of ATRX, recapitulates many human phenotypes. In addition, XNP-1 is required to repress the inappropriate activation of germline genes. Importantly, this germline misexpression correlates with most of the phenotypes observed in xnp-1 animals. Seemingly distinct xnp-1 phenotypes such as developmental abnormalities and telomeric defects are both suppressed by mutation of the germline transcription factor gsox-1 . These findings suggest that the majority of XNP-1-dependent phenotypes stem from its role in maintaining proper cellular identity, offering insights into the functions of ATRX in humans.

The experimental high-throughput screening (HTS) methods, exemplified by CRISPR- based screening techniques, have revolutionized target identification in drug discovery. However, such screens frequently yield extensive, often unrelated target lists necessitating costly and time-intensive experimental evaluation and validation. To address this challenge, we propose a dual-filter strategy that integrates literature-mined targets with CRISPR/Cas9 screening outputs, systematically prioritizing the most credible candidates and thereby reducing the experimental validation burden and increasing success rate. To validate this strategy, we applied it with hand-foot syndrome (HFS), a clinically challenging side effect induced by fluoropyrimidine treatment. We identified ATF4 as a key regulator of 5-fluorouracil (5-FU) toxicity in the skin and revealed forskolin as a potential therapeutic agent of HFS through the strategy. Mechanistically, forskolin triggers MEK/ERK-dependent ATF4 induction, subsequently driving 5-FU detoxification via the ATF4-mediated eIF2α/IκB signaling pathway. Our findings demonstrate that this dual-filter strategy could notably accelerate drug discovery by reducing experimental validation burden after target screening.

Engineered CRISPR gene drives are a promising new strategy for fighting malaria and other vector-borne diseases, made possible by genome engineering with the CRISPR-Cas9 system. One useful approach to predict the outcome of a gene drive mosquito release is individual-based modeling, which can be spatially explicit and allows flexible parameters for drive efficiency, mosquito ecology, and malaria transmission. However, the computational demand of this type of model significantly increases when including a larger number of parameters, especially due to the chasing phenomenon, which can delay or prevent successful population elimination. Thus, we built a simulation-based deep-learning model to comprehensively understand the effects of different parameters on Anopheles gambiae mosquito suppression and malaria prevalence among the human population. The results suggest that reducing the embryo resistance cut rate, reducing the functional resistance forming rate and increasing the drive conversion rate plays the major role in mosquito suppression and related phenomena. We also observed that the parameter space for eliminating malaria was substantially larger than that for mosquito suppression, suggesting that even a considerably imperfect drive may still successfully accomplish its objective despite chasing or resistance allele formation. This study shows that suppression gene drives may be highly effective at locally eliminating malaria, even in challenging conditions.

To date, Leucobacter species have been identified from diverse sources with various ecological and functional roles. However, the genomic features and pathogenic potential of antibiotic-resistant Leucobacter strains remains understudied. Here, we isolated the Leucobacter sp. HNU-1 from tropical Hainan Province, China, and found it can induce diapause in Caenorhabditis elegans following ingestion, while exhibiting no significant effects on the nematode's lifespan, survival rate, locomotion, and intestinal epithelial cells. This bacterium demonstrates resistance to multiple antibiotics, including kanamycin, streptomycin, sulfonamides, and vancomycin. On LB medium, Leucobacter sp. HNU-1 forms yellow, opaque colonies with a smooth, moist surface, regular edges, a convex center, and no surrounding halo, with diameters ranging from 2 to 3 mm. Furthermore, we performed whole-genome sequencing using third-generation high-throughput sequencing technology. De novo assembly revealed a genome size of 3,375,033 bp, with a GC content of 70.37%. A total of 3,270 functional genes, accounting for 88.98% of the genome, were annotated, along with six potential CRISPR sequences and other genetic elements. Genomic and bioinformatic analyses further identified antibiotics-related genes. This research provides a theoretical foundation for investigating antibiotic-resistant environmental bacteria in tropical environments and offers new insights into potential therapeutic strategies for microbial infections and host-microbe interactions.

Summary Although plants share core cell division mechanisms with other eukaryotes, their unique features—such as acentrosomal spindle formation and cytokinesis via the phragmoplast—suggest the existence of plant-specific genes. This study used the model bryophyte Physcomitrium patens to uncover such genes and employed CRISPR-based screening to identify novel cell division genes in plants. Co-expression data from known mitotic genes were used to create a pool of 216 candidate genes, which were then targeted in CRISPR/Cas9 screening. Frameshift mutants with division defects were characterized using high-resolution imaging of mitosis and gene tagging with fluorophores for localization analysis. Three novel gene families—CYR (Cytokinesis-Related), LACH (Lagging Chromosome), and SpinMi (Spindle and Phragmoplast Midzone)—were identified. CYR genes were linked to cytokinesis defects, LACH was essential for chromosome segregation, and SpinMi localized to the spindle and phragmoplast midzone. Notably, none of these gene families had homologs in algae, suggesting their emergence during land colonization. Our findings provide a framework for combining co-expression analysis with targeted screening to identify genes associated with specific cellular processes, in this case, cell division. Beyond characterizing three novel gene families, this study also offers insights into evolutionary changes in the plant cell division machinery.

Collagen VI Related Dystrophies (COL6-RD) are congenital muscle diseases, typically inherited as an autosomal dominant trait. A frequent type of mutation involves glycine substitutions in the triple helical domain of collagen VI alpha chains, exerting a dominant-negative effect on the unaltered protein. Despite this, no prior animal model captured this mutation type. Using CRISPR/Cas9, we generated transgenic mice with the equivalent of the human COL6A1 c.877 G>A; p. Gly293Arg mutation. We characterized their skeletal muscle phenotype over time, utilizing computer-aided tools applied to standardized parameters of muscle pathology and function. Knock-in mice exhibited early-onset reduced muscle weight, myopathic histology, increased fibrosis, reduced collagen VI expression, muscle weakness, and impaired respiratory function. These features provide adequate outcome measures to assess therapeutic interventions. The different automated image analysis methods deployed here analyze thousands of features simultaneously, enhancing accuracy in describing muscle disease models. Overall, the Col6a1 Ki Gly292Arg mouse model offers a robust platform to deepen our understanding of COL6-RD and advance its therapeutic landscape. Summary Statement We generated and characterized over time the first mouse model representing dominant negative glycine substitutions in the alpha chains of collagen VI that are a frequent cause of Collagen VI-Related Dystrophies.

Although GBM’s immunosuppressive environment is well known, the tumor’s resistance to CD8+ T cell killing is not fully understood. Our previous study identified Checkpoint Kinase 2 (Chek2) as the key driver of CD8+ T cell resistance in mouse glioma through an in vivo CRISPR screen and demonstrated that Chk2 inhibition, combined with PD-1/PD-L1 blockade, significantly enhanced CD8+ T cell-mediated tumor killing and improved survival in preclinical model. Here, we aimed to elucidate the immunosuppressive function of Chek2. Immunoprecipitation (IP) followed by mass spectrometry (MS) and phosphoproteomics identified an association between Chek2 with the DNA/RNA-binding proteins YBX1 and YBX3 that are implicated in transcriptional repression of pro-inflammatory genes. Single-gene knock-out and overexpression studies of CHEK2, YBX1, and YBX3 in multiple glioma cell lines revealed that these proteins positively regulate each other’s expression. RNA sequencing coupled with chromatin immunoprecipitation-sequencing (ChIP-seq) analysis demonstrated common inflammatory genes repressed by CHK2-YBX1&YBX3 hub. Targeting one of the hub proteins, YBX1, with the YBX1 inhibitor SU056 led to degradation of CHK2-YBX1&YBX3 hub. Targeting of this hub by SU056 led to enhanced antigen presentation and antigen specific CD8+ T cell proliferation. Further, combination of SU056 with ICB significantly improved survival in multiple glioma models. Collectively, these findings reveal an immunosuppressive mechanism mediated by the CHK2-YBX1&YBX3 hub proteins. Therefore, CHK2-YBX1&YBX3 hub targeting in combination with immune checkpoint blockade therapies in gliomas is warranted.

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 is a conserved feature of RNA polymerase II transcripts 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 phenotype, 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.

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; laboratory model strains as donor versus recent human isolates as donor; and the biofilm substrate utilized 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). We also identified that E. faecalis Δ cas9 has altered biofilm structure and thickness relative to the wild-type strain when cultured on a plastic substrate, but equivalent growth in the agar plate biofilms widely used for conjugation studies. 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.