Single-cell RNA sequencing and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) screening facilitate the high-throughput study of genetic perturbations at a single-cell level. Characterising combinatorial perturbation effects, such as the subset of genes affected by a specific perturbation, is crucial yet computationally challenging in the analysis of single-cell CRISPR screening datasets due to the sparse and complex structure of unknown biological mechanisms. We propose Gaussian process based sparse perturbation regression (GPerturb) to identify and estimate interpretable gene-level perturbation effects for such data. GPerturb uses an additive structure to disentangle perturbation-induced variation from background noise, and can learn sparse, gene-level perturbation-specific effects from either discrete or continuous responses of perturbed samples. Crucially, GPerturb provides uncertainty estimates for both the presence and magnitude of perturbation effects on individual genes. We validate the efficacy of GPerturb on both simulated and real-world datasets, demonstrating that its prediction and generalisation performance is competitive with existing state-of-the-art methods. Using real-world datasets, we also show that the model reveals interesting gene-perturbation interactions and identifies perturbation effects consistent with known biological mechanisms. Our findings confirm the utility of GPerturb in revealing new insights into the complex dependency structure between gene expressions and perturbations.

TDP-43 is an RNA-binding protein constituting the pathological inclusions observed in ∼95% of ALS and ∼50% of FTD patients. In ALS and FTD, TDP-43 mislocalises to the cytoplasm and forms insoluble, hyperphosphorylated and ubiquitinated aggregates that enhance cytotoxicity and contribute to neurodegeneration. Despite its primary role as an RNA/DNA-binding protein, how RNA-binding deficiencies contribute to disease onset and progression is little understood. Among many identified familial mutations in TDP-43 causing ALS/FTD, only two mutations cause an RNA-binding deficiency, K181E and K263E. In this study, we used CRISPR/Cas9 to knock-in the two disease-linked RNA-binding deficient mutations in SH-SY5Y cells, generating both homozygous and heterozygous versions of the mutant TDP-43 to investigate TDP-43-mediated neuronal disruption. Significant changes were identified in the transcriptomic profiles of these cells, in particular, between K181E homozygous and heterozygous cells, with the most affected genes involved in neuronal differentiation and synaptic pathways. This result was validated in cell studies where the neuronal differentiation efficiency and neurite morphology were compromised in TDP-43 cells compared to unmodified control. Interestingly, divergent neuronal regulation was observed in K181E-TDP-43 homozygous and heterozygous cells, suggesting a more complex signalling network associated with TDP-43 genotypes and expression level which warrants further study. Overall, our data using cell models expressing the ALS/FTD disease-causing RNA-binding deficient TDP-43 mutations at endogenous levels show a robust impact on transcriptomic profiles at the whole gene and transcript isoform level that compromise neuronal differentiation and processing, providing further insights on TDP-43-mediated neurodegeneration.

SUMMARY Spiral ganglion neurons (SGNs) are crucial for hearing, and the loss of SGNs causes hearing loss. Stem cell-based therapies offer a promising approach for SGN regeneration and require understanding the mechanisms governing SGN differentiation. We investigated the chromatin remodeler CHD7 in neuronal differentiation using immortalized multipotent otic progenitor (iMOP) cells. We demonstrated that CHD7 knockdown impaired neuronal differentiation. Genome-wide analysis revealed CHD7 binding at diverse cis -regulatory elements, with notable enrichment at sites marked by the insulator-binding protein CTCF between topologically associating domains (TADs). Insulators marked by the enrichment of CHD7 and CTCF resided near genes critical for neuronal differentiation, including Mir9-2 . Targeting these regulatory regions in iMOPs with CRISPR interference (CRISPRi) and activation (CRISPRa) increased miR-9 transcription, irrespective of the method. Blocking the CHD7 and CTCF marked sites suggested that the elements function as insulators to regulate gene expression. The study highlights CHD7 activity at insulators and underscores an unreported mechanism for promoting neuronal differentiation.

ABSTRACT Insulin-like growth factor 1 (IGF1) is produced primarily in the placenta in utero and is an essential hormone for neurodevelopment. Specifically, how placental IGF1 production persistently influences the brain is unclear. This study evaluated the effects of placental Igf1 overexpression on embryonic and postnatal brain development, particularly for striatum, a region highly linked to neurodevelopmental disorders. Placental Igf1 was overexpressed via placental-targeted CRISPR manipulation. This overexpression altered placenta structure and function distinctly in females and males. Early differences in placental function altered the trajectory of striatal development, as adult females showed persistent changes in striatal cell composition and striatal dependent behavior while males were less affected in brain and behavior outcomes. Overall, these results demonstrate that placental Igf1 expression alters striatal development and behavior in ways relevant to neurodevelopmental disorders. These findings expand our understanding of placental influence on neurodevelopment and will aid in identifying placental-targeted preventive interventions. GRAPHICAL ABSTRACT

Droplet-based organoid culture offers several advantages over conventional bulk organoid culture, such as improved yield, reproducibility, and throughput. However, organoids grown in droplets typically display only a spherical geometry and lack the intricate structural complexity found in native tissue. By incorporating singularized pancreatic ductal adenocarcinoma cells into collagen droplets, we achieve the growth of branched structures, indicating a more complex interaction with the surrounding hydrogel. A comparison of organoid growth in droplets of different diameters showed that while geometrical confinement improves organoid homogeneity, it also impairs the formation of more complex organoid morphologies. Thus, only in 750 µ m diameter collagen droplets did we achieve the consistent growth of highly branched structures with a morphology closely resembling the structural complexity achieved in traditional bulk organoid culture. Moreover, our analysis of organoid morphology and transcriptomic data suggests an accelerated maturation of organoids cultured in collagen droplets, highlighting a shift in developmental timing compared to traditional systems.

Cancers, especially fusion oncoprotein (FO)-driven hematological cancers and sarcomas, often develop from a low number of key mutations. Solitary Fibrous Tumor (SFT) is a rare mesenchymal tumor driven by the NAB2-STAT6 oncofusion gene. Currently, the treatment options for SFT remain limited, with anti-angiogenic drugs providing only partial responses and an average survival of two years. To address this challenge, we constructed SFT cell models harboring specific NAB2-STAT6 fusion transcripts using the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) technology. High-throughput drug screens demonstrated that the BET inhibitor Mivebresib can differentially reduce proliferation in SFT cell models. Subsequently, BET inhibitors Mivebresib and BMS-986158 efficiently reduced tumor growth in an SFT patient-derived xenograft (PDX) animal model. Furthermore, our data showed that NAB2-STAT6 fusions may lead to higher levels of DNA damage in SFTs. Consequently, combining BET inhibitors with PARP (Poly (ADP-ribose) polymerase) or ATR inhibitors significantly enhanced anti-proliferative effects in SFT cells. Taken together, our study established BET inhibitors Mivebresib and BMS-986158 as promising anti-SFT agents.

The increasing prevalence of bacteria resistant to many or all types of antibiotics poses a major health crisis. Novel classes of antibiotics are only slowly being developed and alternative strategies are needed to tackle the issue. Mobile genetic elements and class 1 integrons are important facilitators for antibiotic resistance genes, with the latter being highly conserved in human pathogens. The growing prevalence of multidrug-resistant bacteria and the paucity in the development of new antibiotics underscore the urgent need for innovative approaches in the treatment of pathogens. Among these, CRISPR-Cas nucleases can be used to cleave acquired resistance genes, leading to either plasmid curing or cell death if the target is on a chromosome. In this study, we investigate the feasibility of using class 1 integrons as a target for Cas9-based cleavage leading to re-sensitizing antibiotic-resistant bacteria. We analyze the conserved and widespread integrase gene intI1 and conclude that it is a suitable target for Cas-based re-sensitization due to its high sequence conservation and its occurrence largely limited to human pathogens, alleviating the risk of targeting benign bacteria. We developed a broad host range conjugative plasmid encoding a class 1 integron-targeting Cas9 system that leads to removal of resistance plasmids in target bacteria with subsequent re-sensitization towards antibiotics. We find that 290 distinct ARGs co-occur on int1 -harboring plasmids, showing the potential for re-sensitization towards a very broad range of antibiotics.

DNA double-strand breaks (DSBs) pose a critical threat to cellular proliferation and genomic integrity. Upon genotoxic stress, the DNA damage response (DDR) rapidly activates repair pathways and halts cell cycle progression through checkpoint activation. Previously, we demonstrated that DDR-activated c-Abl tyrosine kinase (ABL1) attenuates error-prone late-phase DSB repair. However, the broader functional implications of c-Abl in DDR regulation, and the fate of any residual unrepaired DNA fragments remained poorly understood. Here, we show that c-Abl regulates G2/M checkpoint release by targeting Polo-like kinase1 (Plk1). Depletion or inhibition of c-Abl resulted in increased G2-M accumulation and impaired checkpoint exit. We identified Y217 as a c-Abl phosphorylation site on Plk1, important for Plk1-mediated Claspin destabilization, a key step in G2/M checkpoint release. CRISPR-mediated introduction of the phopsho-silencing Plk1 Y217F mutation or the phospho-mimicking Y217E mutation into cells resulted in impaired and enhanced G2/M checkpoint release, respectively. Intriguingly, c-Abl-mediated G2/M checkpoint release correlated with elevated DNA damage-induced micronuclei (MNi) formation. Depletion or inhibition of c-Abl reduced MNi formation, whereas induction of c-Abl expression increased it, implicating c-Abl as an active effector in both processes. We propose a trade-off model whereby, following the rapid initial repair phase, c-Abl shifts cellular priorities from prolonged, potentially error-prone DSB repair toward cell cycle resumption, thereby promoting G2-M checkpoint exit and DDR deactivation at the cost of increased MNi formation. Our findings describe a novel regulatory DDR axis involving c-Abl and Plk1 and provide mechanistic insights into how DDR termination is orchestrated.

Summary ▪ In strawberry, the axillary bud (AXB) can produce either an elongated stem called stolon giving a daughter-plant (asexual reproduction) or an inflorescence-bearing branch crown (BC) (sexual reproduction). The fate of the AXB depends on node position on the axis and on genetic and environmental factors. Here, in Fragaria vesca , we addressed the largely unanswered question of how molecular factors determine AXB fate. ▪ To get insights into the mechanisms already at play in a morphologically indistinguishable (undifferentiated) AXB, depending on its fate, we combined (1) the phenotypic characterization of AXB development throughout plant growth with (2) the RNA-seq analysis of undifferentiated AXBs, using three different genotypes producing either BCs or stolons ( fvetfl1 and fvega20ox mutants, FveFT3 overexpressor). ▪ Results: allowed the identification of genes regulating AXB fate and outgrowth, among which FveBRC1 . The analysis of FveBRC1 expression in genotypes combining various traits (perpetual/seasonal flowering; runnering/runneless) and the generation of CRISPR/Cas9 brc1 mutants further demonstrated that FveBRC1 plays a central role in the determination of AXB fate in strawberry, in addition to its well-known function in BC outgrowth. ▪ These original results provide new insights into the determination of AXB fate and, consequently, the control of fruit yield in strawberry.

Although lifespan has long been the focus of ageing research, the need to enhance healthspan - the fraction of life spent in good health - is a more pressing societal need. Caloric restriction improves healthspan across eukaryotes but is unrealistic as a societal intervention. Here, we describe the rewiring of a highly conserved nutrient sensing system to prevent senescence onset and declining fitness in budding yeast even when aged on an unrestricted high glucose diet. We show that AMPK activation can prevent the onset of senescence by activating two pathways that remove excess acetyl coenzyme A from the cytoplasm into the mitochondria - the glyoxylate cycle and the carnitine shuttle. However, AMPK represses fatty acid synthesis from acetyl coenzyme A, which is critical for normal cellular function and growth. AMPK activation therefore has positive and negative effects during ageing. Combining AMPK activation with a point mutation in fatty acid synthesis enzyme Acc1 that prevents inhibition by AMPK (the A2A mutant) allows cells to maintain fitness late in life without reducing the mortality associated with advanced age. Our research shows that ageing in yeast is not intrinsically associated with loss of fitness, and that metabolic re-engineering allows high fitness to be preserved to the end of life.

Abstract Background: Engineers seeking to generate natural product analogs through altering modular polyketide synthases (PKSs) face significant challenges when genomically editing large stretches of DNA. Results: We describe a CRISPR-Cas9 system that was employed to reprogram the PKS in Streptomyces venezuelae ATCC 15439 that helps biosynthesize the macrolide antibiotic pikromycin. We first demonstrate its precise editing ability by generating strains that lack megasynthase genes pikAI - pikAIV or the entire pikromycin biosynthetic gene cluster but produce pikromycin upon complementation. We then employ it to replace 4.4-kb modules in the pikromycin synthase with those of other synthases to yield two new macrolide antibiotics with activities similar to pikromycin. Conclusion: Our gene-editing tool has enabled the efficient replacement of extensive and repetitive DNA regions within streptomycetes.

ABSTRACT Deubiquitinases (DUBs) form a specific class of proteases removing ubiquitin from target proteins. They are involved in the regulation of many cellular processes including cell growth and proliferation. Among them, USP36 is a key regulator of the oncogenic transcription factor c-Myc, preventing its degradation by the proteasome. These two proteins form an evolutionary conserved complex providing the opportunity to investigate USP36 mechanisms of action in vivo in a genetically tractable model such as Drosophila melanogaster . Null mutants of dUsp36 die early during larval development and exhibit severe growth defects. Strikingly, we report here that flies carrying a CRISPR/Cas9-induced catalytic mutation of dUsp36 survive to adulthood with only minor growth defects, yet males are infertile. This finding indicates that dUSP36 deubiquitinating activity is dispensable for cell growth but essential for spermatogenesis. Our results thus reveal that dUSP36 functions through both catalytic-dependent and catalytic-independent mechanisms, highlighting a dual mode of action with implications for the understanding of DUBs mechanism of action.

Understanding and manipulating the complexity of microbial community diversity, including the mobile genetic elements contained within, represents a great challenge that has wide-ranging potential benefits across synthetic biology, agriculture and medicine. An important component to this complexity is the acquisition of genetic material via conjugative plasmids, which can encode new functions such as antibiotic resistance, and the degradation of those plasmids by CRISPR interference. In this work we use single-cell tracking of E. coli populations in microfluidic devices coupled with high resolution fluorescence microscopy to characterize plasmid dynamics at the single-cell and population level. On a population level we find that the ability of cells to clear plasmids is highly dependent on both the number of spacer targets present and the defense expression level. Additionally we assess the impact that counter-defense mechanisms such as plasmid addiction have on plasmid population dynamics and show that CRISPR may be an ineffective method to counter plasmids with such mechanisms. By leveraging single-cell tracking, we were also able to report conjugation rate per neighboring donor cell, estimate the latent period between plasmid uptake and subsequent onward transmission, and corroborate existing estimates of cascade search time. This synthesis of population and single-cell measurements suggests that plasmids are the subject of a dynamic tug-of-war between defense expression, spacer distribution, neighboring cell identity and plasmid cost-benefit tradeoffs. The use of imaging and analysis techniques used here and subsequent multi-scale measurements will facilitate the disentanglement of how these factors coordinate to realize community-wide plasmid dynamics in diverse contexts.

CRISPR/Cas mediated transposition is a recently recognized strategy for horizontal gene transfer in a variety of bacterial species. However, our understanding of the factors that control their function in their natural hosts is still limited. In this work we report our initial genetic characterization of the elements associated with the CRISPR/Cas-transposition machinery (CASTm) from Vibrio parahaemolyticus ( Vpa CASTm), which are encoded within the pathogenicity island VpaI-7. Our results revealed that the components of the Vpa CASTm and their associated CRISPR arrays ( Vpa CAST system) are transcriptionally active in their native genetic context. Furthermore, we were able to detect the presence of polycistrons and several internal promoters within the loci that compose the Vpa CAST system. Our results also suggest that the activity of the promoter of the atypical CRISPR array is not repressed by the baseline activity of its known regulator VPA1391 in V. parahaemolyticus . Additionally, we found that the activity of the promoter of tniQ was modulated by a regulatory cascade involving ToxR, LeuO and H-NS. Since it was previously reported that the activity of the Vpa CAST system was less efficient than that of the Vch CAST system at promoting transposition of a miniaturized CRISPR-associated transposon (mini-CAST) in Escherichia coli , we analyzed if the transposition efficiency mediated by the Vpa CAST system could be enhanced inside its natural host V. parahaemolyticus . We provide evidence that this might be the case suggesting that there could be host induction factors in V. parahaemolyticus that could enable more efficient transposition of CASTs. Importance Mobile genetic elements such as transposons play important roles on the evolutionary trajectories of bacterial genomes. The success of transposon dissemination depends on their ability to carry selectable markers that improve the fitness of the host cell or loci with addictive traits such as the toxin-antitoxin systems. Here we aimed to characterize a transposon from Vibrio parahaemolyticus that carries and could disseminate multiple virulence factors. This transposon belongs to a recently discovered family of transposons whose transposition is guided by crRNA. We showed that the transposition machinery of this transposon is transcribed in V. parahaemolyticus and that there are likely host associated factors that favor transposition in the natural host V. parahaemolyticus over transposition in Escherichia coli .

Given the rapid resistance of Plasmodium falciparum to antimalarial drugs, there is a continual need for new treatments. A genome-scale metabolic (GSM) model was developed with integrated omic and constraint-based, experimental flux-balance data to predict genes essential for P. falciparum growth as drug targets. We selected the highly ranked P. falciparum UMP-CMP kinase (UCK) to test its necessity and the ability to inhibit growth with inhibitors. Conditional deletion mutants using the DiCre recombinase system, generated by CRISPR-Cas genome editing, exhibited defective asexual growth and stage-specific developmental arrest. Based on in silico and in vitro screening, inhibitors were identified that are selective for P. falciparum UCK and exhibit antiparasitic activity. This study, for the first time, shows assertions from a GSM model identifying novel, validated, “druggable” targets. These findings show a role for GSM models in antimalarial drug discovery and identify P. falciparum UCK as a novel, valid malaria drug target.

Abstract Leaf rust is a devastating fungal disease of wheat. Planting resistant wheat cultivars is the most effective strategy to mitigate this threat. Here, we generate a 10.51-gigabase chromosome-scale assembly of the durum wheat landrace PI 192051. Using mutagenesis and transcriptome sequencing, we identify the leaf rust resistance gene Lr.ace-4A within a recombination-sparse region of PI 192051 and demonstrate that Lr.ace-4A is identical to the previously designated Lr30 gene in hexaploid wheat. Lr30 / Lr.ace-4A encodes a non-canonical coiled-coil nucleotide-binding leucine-rich repeat receptor, featuring tandem NB-ARC domains. This gene proves both necessary and sufficient to confer resistance to Puccinia triticina , as demonstrated by CRISPR/Cas9-induced mutations and transgenic complementation. Lr30 provides near-immunity resistance in durum wheat, though its effectiveness is diminished in hexaploid wheat. Two amino acid polymorphisms differentiate the resistant and susceptible Lr30 haplotypes, with transgenic plants carrying either variant exhibiting susceptibility. Cloning of Lr30 will accelerate its deployment in wheat breeding programs.

Lack of canonical biomarkers and strategies to target radioresistance contribute to poor patient outcomes in triple-negative breast cancer (TNBC). Identifying and targeting novel radioresistance genes will benefit in enhancing radiotherapy response and treatment outcome in TNBC patients. A genome-wide CRISPR screen was performed to identify radioresistance genes in the MDA-MB-231 TNBC cell line. An in vitro clonogenic assay was used to assess antipro-liferative effects of Artemis knockout or pharmacologic inhibition of Artemis, either alone or in combination with RT. Tumor doubling time and animal survival were assessed using an in vivo xenograft model. RNA-Seq analysis was performed to identify genes and pathways deregulated under Artemis knockout conditions, both alone and in combina-tion with RT. Cellular senescence was evaluated using a -galactosidase assay. Our CRISPR screen identified Artemis as a top hit in RT-treated MDA-MB-231 cells, whose depletion led to radiosensitization in TNBC. Artemis knockout significantly reduced cell proliferation and enhanced the antiproliferative effects of RT in vitro. Compared to mice bearing control MDA-MB-231 xenografts, Artemis knockout exhibited prolonged survival that was further enhanced with RT. Bulk RNA-Sequencing indicated that the antipro-liferative and radiosensitization effects of Artemis depletion were mediated by activation of cellular senescence which was confirmed with a -galactosidase assay. Taken together, our results highlight the critical role of Artemis in TNBC cell proliferation and response to radiation. Our findings identify Artemis as a potential biomarker in-dicative of sensitivity to radiation and a putative target that could be inhibited to enhance the efficacy of RT in TNBC.

Precise coupling between differentiation and metabolism in the kidney proximal tubule (PT) is critical for homeostasis, yet determinants of tubular cell fate specialization remain unclear. Using genetically engineered model organisms, cell cultures, proteomic and metabolomic profiling, and human genetics, we show that ATG7 deficiency shifts PT cell metabolism and differentiation toward growth at the expense of function. ATG7 loss impairs autophagy-mediated lipid droplet homeostasis, causing their accumulation and disrupting fatty acid transfer to mitochondria, compromising energy metabolism and cellular function. In zebrafish, reintroducing human wild-type ATG7 restores homeostasis, whereas inhibiting mitochondrial fatty acid oxidation induces phenotypic changes in wild-type cells. In humans, ATG7 variation associates with increased kidney disease risk, and loss-of-function variants cause tubular proteinuria and altered mitochondrial metabolism. Low ATG7 levels correlate with dedifferentiation, altered metabolic pathways, and poor renal cell carcinoma outcomes. Together, our results establish a metabolic paradigm that links autophagy to kidney epithelial cell fate specialization, with broad implications for health and disease.

Inland recirculating aquaculture is a thriving food industry providing sustainable and locally sourced high-quality protein. However, its expansion is accompanied by emerging challenges regarding the spread of pathogens and diseases. Detection and management of pathogens in aquaculture remain underdeveloped compared to other animal farming sectors due to the vast diversity of species involved, the non-domesticated species, and limited knowledge regarding pathogens, host responses, and disease mechanisms. Furthermore, recirculating aquaculture systems are heavily dependent on beneficial bacterial communities for waste product removal and water quality maintenance, with opportunistic pathogens constituting an inherent component of these microbial communities. To enhance the potential of inland aquaculture as a sustainable source of protein, it is imperative to adopt advanced tools for pathogen detection and monitoring and for assessing the overall health of the microbial ecosystem. This paper presents an overview of promising current molecular and technological advancements that offer solutions for pathogen detection and system monitoring in aquaculture. We focus on recently developed point-of-care and on-site detection methods using miniaturized laboratory equipment and robust workflows that operate independently of cold chain logistics. We explore current methodologies for monitoring pathogens in the environment rather than through fish health assessments. Lastly, we discuss techniques from other scientific disciplines in aquaculture, including CRISPR-Cas protocols for pathogen detection and the implementation of "omics" approaches for comprehensive characterization of microbial states. These methods demonstrate considerable potential for pathogen surveillance and, subsequently, early responses in the dynamic aquaculture field. Through a better understanding of available options aquaculture managers and molecular scientist can collaborate and optimize systems. This paper aims to facilitate communication between molecular scientists and aquaculture managers, equipping the aquaculture industry with knowledge to enhance pathogen management techniques in their facilities.

CRISPR/Cas9 (clustered regulatory interspaced short palindromic repeats/CRISPR-associated 9) has revolutionized gene editing technology by offering high precision and efficiency. Despite its powerful potential, significant challenges remain to completely avoid side effects and to improve delivery systems. This review focuses on CRISPR/Cas9 system delivery cargoes and on the different types of delivery vehicles. Emphasis is placed on advantages, disadvantages and gene editing efficiency of each method as well as introducing delivery vehicles such as lipid nanoparticles, which offer potential to enhance precision and reduce immune responses. Additionally, Cas9 aggregation behavior, a factor rarely described in literature, is reviewed due to its potential impact on editing efficiency, an often-overlooked factor that could influence future therapeutic applications. By addressing these challenges, gene editing moves closer to maximizing its therapeutical applications.

Respiratory syncytial virus (RSV) is a globally prevalent pathogen, causes severe disease in older adults, and is the leading cause of bronchiolitis and pneumonia in the United States for children during their first year of life [1]. Despite its prevalence worldwide, RSV-specific treatments remain unavailable for most infected patients. Here, we leveraged a combination of genome-wide CRISPR knockout screening and single-cell RNA sequencing to improve our understanding of the host determinants of RSV infection and the host response in both infected cells, and uninfected bystanders. These data reveal temporal transcriptional patterns that are markedly different between RSV infected and bystander activated cells. Our data show that expression of interferon-stimulated genes is primarily observed in bystander activated cells, while genes implicated in the unfolded protein response and cellular stress are upregulated specifically in RSV infected cells. Furthermore, genome-wide CRISPR screens identified multiple host factors important for viral infection, findings which we contextualize relative to 29 previously published screens across 17 additional viruses. These unique data complement and extend prior studies that investigate the proinflammatory response to RSV infection, and juxtaposed to other viral infections, provide a rich resource for further hypothesis testing. Importance Respiratory syncytial virus (RSV) is a leading cause of lower respiratory tract infection in infants and the elderly. Despite its substantial global health burden, RSV-targeted treatments remain unavailable for the majority of individuals. While vaccine development is underway, a detailed understanding of the host response to RSV infection and identification of required human host factors for RSV may provide insight into combatting this pathogen. Here, we utilized single-cell RNA sequencing and functional genomics to understand the host response in both RSV infected and bystander cells, identify what host factors mediate infection, and contextualize these findings relative to dozens of previously reported screens across 17 additional viruses.

ABSTRACT CRISPR-Cas12a enzymes are versatile RNA-guided genome-editing tools with applications encompassing viral diagnosis, agriculture and human therapeutics. However, their dependence on a 5’-TTTV-3’ protospacer-adjacent motif (PAM) next to DNA target sequences restricts Cas12a’s gene targeting capability to only ∼1% of a typical genome. To mitigate this constraint, we used a bacterial-based directed evolution assay combined with rational engineering to identify variants of Lachnospiraceae bacterium Cas12a (LbCas12a) with expanded PAM recognition. The resulting Cas12a variants use a range of non-canonical PAMs while retaining recognition of the canonical 5’-TTTV-3’ PAM. In particular, biochemical and cell-based assays show that the variant Flex-Cas12a utilizes 5’-NYHV-3’ PAMs that expand DNA recognition sites to ∼25% of the human genome. With enhanced targeting versatility, Flex-Cas12a unlocks access to previously inaccessible genomic loci, providing new opportunities for both therapeutic and agricultural genome engineering.

DNA plasmids (pDNA) are essential for gene cloning and protein expression, whereby engineered plasmids serve as vectors to insert foreign DNA into host cells, enabling mass production of proteins and vaccines. Furthermore, pDNA is used in CRISPR-based gene editing, RNA therapeutics, and DNA vaccines. Due to the rapidly increasing use and application of a wide variety of pDNA, analytical methods to characterize their key attributes are vital. Because of their high molecular weight, accurate and fast mass analyses of pDNA, as a measure of quality control, is rather challenging. Here we explore mass photometry (MP) to analyze pDNAs and find that it completely fails using standard procedures as developed for MP on proteins, with masses underestimated by 30-40%. Even though the landing of pDNA during MP analysis can be improved by using coated glass slides, the large dsDNA particles diffract light beyond the diffraction limit, rendering most landing events unusable. To overcome these issues, we introduce a fast (30 s) and simple protocol to convert dsDNA particles rapidly into ssDNA-like particles just prior to analysis and show that these particles behave nearly perfect for MP. Using this protocol accurate and correct masses of pDNAs can be obtained by MP, with values within 1-3% of the expected mass. Using this protocol, MP can be used to mass analyze pDNA constructs from 1 to 15 MDa, suggesting that this approach may be widely adopted within academia and biopharma for essentially all plasmids.

Intronic hexanucleotide repeat expansions in the C9orf72 gene represent the most common genetic cause of the neurodegenerative diseases amyotrophic lateral sclerosis (ALS) and frontotemporal dementia. This expansion decreases C9orf72 expression in affected patients, indicating that loss of C9orf72 function (LOF) acts as a pathogenic mechanism. Several models using Danio rerio (zebrafish) for C9orf72 depletion have been developed to explore disease mechanisms and the consequences of C9orf72 LOF. However, inconsistencies exist in reported phenotypes, and many have yet to be validated in stable germline ablation models. To address this, we created a zebrafish C9orf72 knockout model using CRISPR/Cas9. The C9orf72 LOF model demonstrates, in a generally dose-dependent manner, increased larval mortality, persistent growth reduction, and motor deficits. Additionally, homozygous C9orf72 LOF larvae exhibited mild overbranching of spinal motoneurons. To identify potential therapeutic compounds, we performed a screen on an established Caenorhabditis elegans ( C. elegans ) C9orf72 homologue ( alfa-1 ) LOF model, identifying 12 compounds that enhanced motility, reduced neurodegeneration, and alleviated paralysis phenotypes. Motivated by the shared motor phenotype, 2 of those compounds were tested in our zebrafish C9orf72 LOF model. Pizotifen malate was found to significantly improve motor deficits in C9orf72 LOF zebrafish larvae. We introduce a novel zebrafish C9orf72 knockout model that exhibits phenotypic differences from depletion models, providing a valuable tool for in vivo C9orf72 research and ALS therapeutic validation. Furthermore, we identify pizotifen malate as a promising compound for further preclinical evaluation. Author Summary Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder characterized by the progressive loss of motor neurons, with no curative treatments currently available. The most common genetic cause is a hexanucleotide repeat expansion in the C9orf72 gene, which reduces its expression and implicates loss-of-function (LOF) as a disease mechanism. However, the complete functions of C9orf72 and its role in ALS remain unclear. Zebrafish models with indirect partial reduction of C9orf72 expression have shown promise in recapitulating key aspects of ALS, but inconsistencies have been observed across these models. To address these challenges, we developed a stable genetic C9orf72 LOF zebrafish model to study the effects of its LOF, validate previous findings, and test potential ALS therapeutics. Our model displays swimming activity deficits, reduced growth, increased mortality, and mild spinal motor neuron abnormalities. We demonstrated that pizotifen malate significantly improved motor function in both our model and a similar well-established worm model. These results underscore the differences between indirect depletion and direct genetic LOF models while identifying pizotifen malate as a promising candidate for preclinical testing. This zebrafish model serves as a valuable tool for understanding C9orf72 -associated ALS mechanisms and advancing therapeutic development.

Abstract RNA displays significant heterogeneity in its structures, dynamics, and functional roles within cells. CRISPR-based imaging methods provide a powerful approach to visualize RNA molecules in living cells. However, the expression of constitutive fluorescence modules in traditional CRISPR-based tools always produce high background and nonspecific signals. To address these challenges, we developed a switched fluorescent CRISPR-tDeg (CtDeg) system that minimizes background for RNA imaging in living cells. This CtDeg system consists of a signal module-deactivated Cas13 protein (dCas13)-degron complex and an engineered switched sgRNA. The signal module-fused complex is degraded unless it binds to the engineered sgRNA, which is activated upon targeting the desired RNA. This interaction will generate a stable signal module-dCas13: sgRNA complex, enabling specific imaging of RNA in living cells. Using the CtDeg system, we visualized the movement and assembly dynamics of paraspeckles at the RNA level. Additionally, we tracked SARS-CoV-2 genome dynamics at the early stage after viral invasion for the first time, providing direct imaging data that demonstrate SARS-CoV-2 infection up-regulates the expression levels of the disease-associated NEAT1 gene, thereby filling a gap in this research area. The CtDeg system represents a powerful tool for specific imaging of RNA to reveal the underlying functions and interactions within living cells.