Pennycress ( Thlaspi arvense ) is being developed as a winter annual intermediate oilseed bioenergy crop in the Midwest during typical fallow periods. Crucial work remains to domesticate and optimize pennycress for incorporation into cropping systems and increasing resilience to rising temperatures. We found that increased planting density reduces biomass and hastens time to flowering and maturity, which are associated with shade avoidance responses. In controlled conditions, we found that pennycress elongates in response to foliar shade and increased ambient temperatures (28 °C). We applied the knowledge base from Arabidopsis thaliana to manipulate genes in the PHYB signaling pathway to simultaneously decrease the shade avoidance response during interseeding and tissue responses to elevated temperatures. Evaluation of CRISPR alleles of PIF7 shows that pif7 reduces organ elongation to competition and heat cues and retains a compact rosette when exposed to shade or elevated temperature and their combination. Crucially, yield and oil content were unaltered in pif7 and plants maintained earlier flowering in stress conditions. Furthermore, indicators of plant health, such as hue, chlorophyll indices, and root system architecture, were improved between wild type and pif7 . This is evidence that plant architecture and physiological health can be uncoupled under competition and heat conditions, supporting our efforts to attenuate morphological responses to environmental cues. We propose this strategy for reducing SAR, improving pennycress performance at high densities, for during interseeding establishment in standing crops, and in a warming climate.

RNA alternative splicing is a fundamental cellular process implicated in cancer development. Kaposi’s sarcoma-associated herpesvirus (KSHV), the etiological agent of multiple human malignancies, including Kaposi’s sarcoma (KS), remains a significant concern, particularly in AIDS patients. A CRISPR-Cas9 screening of matched primary rat mesenchymal stem cells (MM) and KSHV-transformed MM cells (KMM) identified key splicing factors involved in KSHV-induced cellular transformation. To elucidate the mechanisms by which KSHV-driven splicing reprogramming mediates cellular transformation, we performed transcriptomic sequencing, identifying 131 differential alternative splicing transcripts, with exon skipping as the predominant event. Notably, these transcripts were enriched in vascular permeability, multiple metabolic pathways and ERK1/2 signaling cascades, which play key roles in KSHV-induced oncogenesis. Further analyses of cells infected with KSHV mutants lacking latent genes including vFLIP, vCyclin and viral miRNAs, as well as cells overexpressing LANA, revealed their involvement in alternative splicing regulation. Among the identified splicing factors, FAM50A, a component of the spliceosome complex C, was found to be crucial for KSHV-mediated transformation. FAM50A knockout resulted in distinct splicing profiles in both MM and KMM cells, and significantly inhibited KSHV-driven proliferation, cellular transformation and tumorigenesis. Mechanistically, FAM50A knockout altered SHP2 splicing, promoting an isoform with enhanced enzymatic activity that led to reduced STAT3 Y705 phosphorylation in KMM cells. These findings reveal a novel paradigm in which KSHV hijacks host splicing machinery, specifically FAM50A-mediated SHP2 splicing, to sustain STAT3 activation and drive oncogenic transformation. Importance Kaposi’s sarcoma-associated herpesvirus (KSHV) causes cancers such as Kaposi’s sarcoma, particularly in AIDS patients. This study uncovers how KSHV hijacks a fundamental cellular process called RNA splicing to promote cancer development. We identified key splicing events that alter critical pathways involved in vascular permeability, metabolism, and oncogenic signaling, particularly ERK1/2 and STAT3. A specific protein, FAM50A, was found to be essential for KSHV-driven cancerous transformation. Removing FAM50A disrupted splicing, weakening cancer-promoting signals. These findings provide new insights into how viruses manipulate host cells to drive cancer and highlight RNA splicing as a potential target for future therapies.

ABSTRACT The rise of difficult-to-treat Mycobacterium abscessus infections presents a growing clinical challenge due to the immense arsenal of intrinsic, inducible and acquired antibiotic resistance mechanisms that render many existing antibiotics ineffective against this pathogen. Moreover, the limited success in discovery of novel compounds that inhibit novel pathways underscores the need for innovative drug discovery strategies. Here, we report a strategic advancement in PROSPECT (PRimary screening Of Strains to Prioritize Expanded Chemistry and Targets), which is an antimicrobial discovery strategy that measures chemical-genetic interactions between small molecules and a pool of bacterial mutants, each depleted of a different essential protein target, to identify whole-cell active compounds with high sensitivity. Applying this modified strategy to M. abscessus , in contrast to previously described versions of PROSPECT which utilized protein degradation or promoter replacement strategies for generating engineered hypomorphic strains, here we leveraged CRISPR interference (CRISPRi) to more efficiently generate mutants each depleted of a different essential gene involved in cell wall synthesis or located at the bacterial surface. We applied this platform to perform a pooled PROSPECT pilot screen of a library of 809 compounds using CRISPRi guides as mutant barcodes. We identified a range of active hits, including compounds targeting InhA, a well-known mycobacterial target but under-explored in the M. abscessus space. The unexpected susceptibility to isoniazid, traditionally considered to be ineffective in M. abscessus , suggested a complex interplay of several intrinsic resistance mechanisms. While further complementary efforts will be needed to change the landscape of therapeutic options for M. abscessus , we propose that PROSPECT with CRISPRi engineering provides an increasingly accessible, high-throughput target-based phenotypic screening platform and thus represents an important step towards accelerating early-stage drug discovery.

ABSTRACT Considerable offseason farmland lays fallow because there are few crops that can profitably fit between primary crops. To remedy, we employed CRISPR genome editing to the freeze-tolerant, rapid cycling wild Brassica, Thlaspi arvense L. (field pennycress). High-yielding domesticated pennycress varieties were created having seed compositions comparable to “double low” canola (low erucic acid and glucosinolate). Seed glucosinolate content was reduced 75 % by combining mutations in R2R3-MYB (MYB28) and bHLH MYC (MYC3) transcription factors. Pennycress weediness was greatly reduced by knockout of the bHLH transcription factor TRANSPARENT TESTA8 ( TT8 ), lowering seed dormancy and seed coat protections thereby mitigating pennycress re-emergence in fields. Domesticated pennycress offers farmers a profitable, low-carbon-intensity intermediate crop that confers ecosystem benefits while producing grain for renewable fuels and enhanced food security.

ABSTRACT Triple-negative breast cancer (TNBC) is an aggressive subtype with poor prognosis and limited treatments. Although 40-70% of TNBC cases overexpress EGFR, anti-EGFR therapies show minimal clinical benefit. This may result from inherent resistance, inactive EGFR, or its absence on the plasma membrane. Here, we used genome wide CRISPR knock out library screening to identify factors that mediate resistance to EGFR inhibitor. We discovered that depletion of a redox protein, thioredoxin reductase 3 (TXNRD3), in MDA-MB-231 cells reduces cell survival after Erlotinib treatment suggesting that loss of TXNRD3 may sensitize TNBC cells to EGFR inhibitors. siRNA-induced knockdown or pharmacological inhibition of TXNRD3 using an FDA-approved drug Auranofin significantly sensitized EGFR-high TNBC cells to EGFR inhibitors. Mechanistically, TXNRD3 knockdown or inhibition using Auranofin increased oxidative stress-mediated accumulation of phosphorylated EGFR at Y1068 and increased surface accumulation. Interestingly, combination of Auranofin with EGFR inhibition markedly induced antibody-dependent cell mediated cytotoxicity (ADCC) and exerted a significant anti-cancer activity in vivo model. Overall, our findings indicate that targeting TXNRD3 with Auranofin can activate EGFR and enhance its surface localization, thereby sensitizing TNBC cells to anti-EGFR therapies. This approach offers a promising strategy for treating TNBC patients who are resistant to current EGFR-targeted treatments.

Clownfish exhibit striking color patterns, characterized primarily by the presence of zero to three vertical white bars, along with three main colors: orange, white, and black. The common ancestor of clownfish likely possessed three vertical bars, with several instances of gains and losses occurring throughout clownfish evolutionary history over the past 10 million years. However, the evolutionary genomic mechanisms underlying the gain or loss of vertical bars remain unknown. In this study, we tested whether vertical bar transitions across the clownfish phylogeny were associated with changes in non-synonymous to synonymous substitution rates ( d N /d S values). Our analyses identified pigmentation-related genes that underwent changes in selective pressure, including gch2, oca2 , and vps11 , which are linked to melanophores, iridophores, and visual function. Additionally, pmel , a key melanogenesis gene, was found under positive selection, suggesting its role in shaping bar patterning. These results provide new insights into the genomic basis of coloration in clownfish, highlighting how selection and genetic variation influence phenotypic evolution.

Small G protein ARL13B localizes to the cilium and plays essential roles in cilium biogenesis, organization, trafficking, and signaling. Here, we established multiple ARL13B knockout cell lines using the CRISPR/Cas9 system. Surprisingly, all our cell lines lost their cilia completely, in contrast to the reported short cilium and reduced ciliogenesis phenotype. We found that multiple regions of ARL13B are necessary for a complete rescue. Additionally, we found that ARL13B knockout cells also lost their response to sonic hedgehog stimulation. Our work demonstrates the absolute requirement of ARL13B for ciliogenesis and sonic hedgehog signaling, at least in cultured cells, and suggest that ARL13B plays a far more critical role in ciliary function than previously understood.

ABSTRACT The mechanical properties of cells are dynamic, allowing them to adjust to different needs in different biological contexts. In recent years, advanced biophysical techniques have enabled the rapid, high-throughput assessment of single-cell mechanics, providing new insights into the regulation of the mechanical cell phenotype. However, the molecular mechanisms by which cells maintain and regulate their mechanical properties remain poorly understood. Here, we present a genome-scale RNA interference (RNAi) screen investigating the roles of kinase and phosphatase genes in regulating single-cell mechanics using Real-Time Fluorescence and Deformability Cytometry (RT-FDC). Our screen identified 82 known and novel mechanical regulators across diverse cellular functions from 214 targeted genes, leveraging RT-FDC’s unique capabilities for comprehensive, high-throughput mechanical phenotyping with single-cell and cell cycle resolution. These findings refine our understanding of how signaling pathways coordinate structural determinants of cell mechanical phenotypes and provide a starting point for uncovering new molecular targets involved in biomechanical regulation across diverse biological systems. SIGNIFICANCE Cell mechanical properties are tightly regulated and play pivotal roles in processes ranging from tissue morphogenesis to disease progression. Despite their importance, the genetic regulation of single-cell mechanics remains largely unexplored. This study represents one of only a few large-scale mechanomic investigations conducted to date. It is the first study to leverage RT-FDC’s unique capability for high-throughput mechanical phenotyping with single-cell and cell cycle resolution to detect gene impacts that may be overlooked in lower-throughput or population-level studies. The mechanical genes identified here provide valuable data points for understanding how cells control their mechanical state and serve as a foundation for future studies exploring the molecular basis of biomechanical regulation.

The dark proteome includes a rapidly expanding catalog of microproteins with unknown functions that have been historically ignored in genome annotations. Here, we exploit an in vivo single-cell CRISPR screening strategy in the mouse epidermis to systematically investigate the tissue-wide function of microproteins. We document the global and cell-type-specific roles of microproteins during epidermal development and homeostasis at single-cell transcriptomic resolution. Focusing on select candidates, we identify a novel microprotein on Gm10076 , identical to the ribosomal intersubunit bridge protein RPL41, whose perturbation strongly impairs proliferation and protein synthesis. Employing ribosome profiling and RNA sequencing, we show that Gm10076 perturbation profoundly reshapes the translational landscape. Contrary to its prior classification as nonessential, we find that the ribosomal protein RPL41 is essential for cellular proliferation, warranting further investigation into its role as an intersubunit bridge in the translational machinery. Together, our study comprehensively charts the tissue-wide functional landscape of the dark proteome, uncovers a second Rpl41 gene critical for ribosome function and establishes a basis for exploring the impact of microproteins on disease pathogenesis.

Obesity is a major public health crisis, affecting billions worldwide and increasing the risk of metabolic and cardiovascular diseases. While lifestyle factors play a role, genetic variation is a key determinant of both obesity susceptibility and the efficacy of treatment strategies. Recent studies have implicated the Semaphorin 3 signalling pathway in obesity; however, specific roles for pathway components remain largely unexplored. Here, we focus on Class A Plexins and their potential contributions to body weight regulation. Using large-scale genetic association data, we identified that rare, predicted loss-of-function mutations in PLXNA4 were associated with body mass index (BMI) in females. Furthermore, common variant analysis revealed that genetic variation at PLXNA4 was linked to BMI, height, and various neuropsychiatric disorders. To investigate the biological role of Plxna4, we generated zebrafish plxna4 loss-of-function mutants, which exhibited an 85–92% reduction in Plxna4 protein. Despite appearing morphologically normal, mutant zebrafish at juvenile stages were shorter, had increased body fat levels relative to size-matched wild-type siblings, and displayed hypertrophic subcutaneous adipose tissue. Feeding assays revealed that plxna4 mutants consumed more food than wild-type siblings and exhibited food-stimulated hyperactivity, characterised by increased swimming speed, higher speed variability, and frequent high-speed bursts. Together, these findings demonstrate a conserved role for Plxna4 in regulating feeding behaviour and body fat levels, providing new insights into the genetic basis of obesity and warranting further studies to elucidate the molecular mechanisms underlying these effects.

Genetic engineering is a formidable approach to study biology. The development of CRISPR-Cas9 has allowed the genetic engineering of insect species from several orders, and in some species, this tool is used routinely for genetic research. However, insect gene editing often relies on the delivery of CRISPR-Cas9 components via embryo injection. This technique has a limitation: some species lay their eggs inside hard substrates or living hosts, making embryo collection impossible or labour-intensive. Recently, a variety of techniques that exploit maternal injection of nucleases have been developed to circumvent embryo injection. Yet, despite this variety of maternal delivery techniques, some insects remain refractory to gene editing. One of these is the parasitoid wasp, Nasonia vitripennis , an important hymenopteran model species. In this study, a recently developed method termed SYNCAS was used to perform knock-out (KO) of the cinnabar gene in this wasp, obtaining KO efficiencies up to ten times higher than reported for other maternal injection approaches. We found up to 2.73% of all offspring to display a KO phenotype, and we obtained up to 68 KO offspring per 100 injected mothers. The optimal timing of injection and provision of hosts for egg laying was determined. With this protocol, routine applications of CRISPR-Cas9 become feasible in this species, allowing reverse genetics studies of genes with unknown associated phenotypes and paving the way for more advanced editing techniques. Abstract Figure Maternal injection of Cas9 RNP together with BAPC and saponins (SYNCAS) results in efficient gene editing of Nasonia vitripennis offspring Timing of injection and egg collection is important for maximising efficiency and reducing screening effort The efficient genetic engineering of Nasonia vitripennis suggests the applicability of SYNCAS to other parasitoid wasps, a category of insects particularly difficult to genetically engineer with embryonic Cas9 injections.

CRISPR/dCas9-based epigenome editing systems, including DNA methylation epimodifiers, have greatly advanced molecular functional studies revolutionizing their precision and applicability. Despite their promise, challenges such as the magnitude and stability of the on-target editing and unwanted off-target effects underscore the need for improved tool characterization and design. We systematically compared specific targeting of the BACH2 gene promoter and genome-wide off-target effects of available and novel dCas9-based DNA methylation editing tools over time. We demonstrate that multimerization of the catalytic domain of DNA methyltransferase 3A enhances editing potency but also induces widespread, early methylation deposition at low-to-medium methylated promoter-related regions with specific gRNAs and, interestingly, also with non-targeting gRNAs. A small fraction of the methylation changes associated with transcriptional dysregulation and mapped predominantly to bivalent chromatin associating both with transcriptional repression and activation. Additionally, specific non-targeting control gRNA caused pervasive and long-lasting methylation-independent transcriptional alterations particularly in genes linked to RNA and energy metabolism. CRISPRoff emerged as the most efficient tool for stable targeting of the BACH2 promoter, with fewer and less stable off-target effects compared to other epimodifiers but with persistent transcriptome alterations. Our findings highlight the delicate balance between potency and specificity of epigenome editing and provide critical insights into the design and application of future tools to improve their precision and minimize unintended consequences.

Sulfolobus islandicus , an emerging crenarchaeal model organism, offers unique advantages for metabolic engineering and synthetic biology applications owing to its ability to thrive in extreme environments. Although several genetic tools have been established for this organism, the lack of well-characterized chromosomal integration sites has considerably limited its potential as a cellular factory. Here, we systematically identified and characterized 13 artificial CRISPR RNAs (crRNAs) targeting eight chromosomal integration sites in S. islandicus using the CRISPR-COPIES pipeline and a multi-omics-informed computational workflow. By leveraging the endogenous CRISPR-Cas systems, we integrated the reporter gene lacS into these sites and validated heterologous gene expression through a β -galactosidase reporter assay, which revealed significant positional effects on expression levels. As a proof of concept, we utilized these characterized sites to genetically manipulate lipid ether composition by overexpressing GDGT (glycerol dibiphytanyl glycerol tetraether) ring synthase B (GrsB) in S. islandicus , a key enzyme in GDGT biosynthesis. This study expands the genetic toolbox for S. islandicus and highlights a concept that could be widely applicable to other Sulfolobales , advancing their potential as robust platforms for archaeal synthetic biology and industrial biotechnology.

Folate metabolism is intricately linked to purine de novo synthesis through the incorporation of folate-derived one-carbon units into the purine scaffold. Here, we investigate the chemical and genetic dependencies caused by mutations in the folate enzyme MTHFD1 and discover a key role for Nudix hydrolase 5 (NUDT5) in regulating purine de novo synthesis. Through genetic knockout and development of a selective chemical NUDT5 degrader, we uncover an unprecedented scaffolding role rather than NUDT5 enzymatic activity is responsible for this phenotype. We find that NUDT5 interacts with the rate-limiting enzyme of purine de novo synthesis, PPAT, to repress the pathway in response to elevated purine levels. Our findings establish NUDT5 as an important regulator of purine de novo synthesis and elucidate its role in mediating sensitivities to 6-thioguanine in cancer treatment and to adenosine in MTHFD1 deficiency.

Summary Most eukaryotic proteins assemble into multisubunit complexes that coordinate essential cellular functions, yet the principles governing their assembly and proteostatic control remain largely undefined. Here, we systematically dissect the cellular assembly and functional organization of the RNA exosome, an essential ribonucleolytic complex, using an inducible dual-guide CRISPR/Cas9 system in mouse embryonic stem cells. We reveal a sequential assembly pathway where Exosc2, Exosc4, and Exosc7 initiate complex formation, facilitating the incorporation of barrel and cap subunits in a defined hierarchy. Unlike other structural subunits, the terminally incorporated cap subunit Exosc1 is dispensable for cell viability, revealing a modular, functionally resilient architecture. We demonstrate that orphan subunits are selectively degraded via the ubiquitin-proteasome system, enforcing stringent quality control over RNA exosome biogenesis. These findings establish a framework for decoding the assembly logic of essential macromolecular machines and uncover previously unrecognized plasticity in the composition and function of the RNA exosome.

Transposable elements are abundant in host genomes but are generally considered to be confined to the cell in which they are expressed, with the notable exception of endogenous retroviruses. Here, we identify a group of LTR retrotransposons that infect the germline from somatic cells within the Drosophila ovary, despite lacking the fusogenic Envelope protein typically required for retroviral entry. Instead, these elements encode a short transmembrane protein, sORF2, with structural features reminiscent of viral cell-cell fusogens. Through genetics, imaging, and electron microscopy, we show that sORF2 localizes to invasive somatic protrusions, enabling the direct transfer of retrotransposon capsids into the oocyte. Remarkably, sORF2-like proteins are widespread among insect retrotransposons and also occur in piscine nackednaviruses and avian picornaviruses. These findings reveal a noncanonical, Envelope-independent transmission mechanism shared by retrotransposons and non-enveloped viruses, offering important insights into host-pathogen evolution and soma-germline interactions.

Scaffold proteins play crucial roles in subcellular organization and function. In many organisms, proteins with multiple Tudor domains are required for the assembly of membraneless RNA-protein organelles (germ granules) in germ cells. Tudor domains are protein-protein interaction modules which bind to methylated polypeptides. Drosophila Tudor protein contains eleven Tudor domains, which is the highest number known in a single protein. The role of each of these domains in germ cell formation has not been systematically tested and it is not clear if some domains are functionally redundant. Using CRISPR methodology, we generated mutations in several uncharacterized Tudor domains and showed that they all caused defects in germ cell formation. Mutations in individual domains affected Tudor protein differently causing reduction in protein levels, defects in subcellular localization and in the assembly of germ granules. Our data suggest that multiple domains of Tudor protein are all needed for efficient germ cell formation highlighting the rational for keeping many Tudor domains in protein scaffolds of biomolecular condensates in Drosophila and other organisms.

Background Phosphoproteomics studies in both cultured and native collecting duct (CD) cells showed that vasopressin strongly increases protein kinase A (PKA)-dependent phosphorylation of β-catenin at Ser552. Relatively little is known about the role of Ser552 phosphorylation. Methods To address the role of β-catenin Ser552 phosphorylation in the mature renal CD, we have inserted a Ser552Ala mutation in mice using CRISPR-Cas9. Results The mutation did not affect the renal abundance of the vasopressin-regulated water channel aquaporin-2 or urinary osmolality. However, the structure of the CD system was altered. Specifically, the cortical branching ratio (the number of nephrons that merge to form one cortical CD) was reduced from 6.18 ± 0.66 in control mice to 3.33 ± 0.82 in Ser552Ala mice. This was associated with a greater number of cortical and medullary CDs with smaller average diameter. The total number of nephrons (glomerular counts) was not different between wild-type and Ser552Ala mice (both ~13,500 per kidney). RNA-seq in microdissected cortical CDs of the mice revealed a highly significant enrichment of genes involved in regulation of mitosis and the cell cycle, along with decreases in mRNAs coding for two cyclin-dependent kinase inhibitor proteins, Cdkn1b and Cdkn1c . At the same time, there were no changes in abundances of major transporter mRNAs, indicative of sustained CD differentiation. A subset of cortical CD cells showed an increase in DNA content, consistent with G2/M cell-cycle arrest. Conclusions The observed structural changes in the collecting duct system of adult mice point to a role of vasopressin-mediated post-translational modification of β-catenin at Ser552 in collecting duct development, presumably PKA-mediated Ser552 phosphorylation. We speculate that vasopressin may act to slow or halt branching morphogenesis perinatally and may affect the collecting duct elongation process that normally produces the unbranched region of the CD system in the cortex and outer medulla. Key points Vasopressin regulates collecting duct (CD) transport by triggering phosphorylation of multiple proteins including β-catenin at Ser552. Mutating Ser552 to a non-phosphorylatable amino acid in mice resulted in altered CD branching without loss of differentiation in adult CDs. The findings point to a role for β-catenin Ser552 phosphorylation in CD branching and sub-segmental CD elongation.

Animal African trypanosomosis (AAT), caused by protist parasites of the genus Trypanosoma, puts upward of a million head of livestock at risk across 37 countries in Africa. The economic impact of AAT and the presence of human-infectious trypanosomes in animals place a clear importance on improving diagnostics for animal trypanosomes to map the distribution of the veterinary parasites and identify reservoirs of human-infectious trypanosomes. We have adapted the CRISPR-based detection toolkit SHERLOCK (Specific High-sensitivity Enzymatic Reporter unLOCKing) for trypanosomatid parasites responsible for AAT (SHERLOCK4AAT) including Pan- trypanosomatid, Trypanozoon , T. vivax , T. congolense , T. theileri , T. simiae and T. suis assays. To test the applicability of this technique in the field, we analysed dried blood spots collected from 200 farm and 224 free-ranging pigs in endemic and historical human African trypanosomiasis foci in Guinea and Côte d’Ivoire, respectively. The results revealed that SHERLOCK4AAT can detect and discriminate between trypanosome species involved in multiple infections with a high sensitivity. 62.7 % [58.1, 67.3] of pigs were found infected with at least one trypanosome species. T. brucei gambiense , a human-infectious trypanosome, was found in one animal at both sites, highlighting the risk that these animals may act as persistent reservoirs. These data suggest that, due to their proximity to humans and their attractiveness to tsetse flies, pigs could act as sentinels to monitor T. b. gambiense circulation using the SHERLOCK4AAT toolbox.

Genome editing technologies create the potential for genetic studies and innovative gene therapies. Here we present new CRISPR-Cas9 tools, named ZIP editors (ZE), loaded with a single-stranded oligodeoxynucleotide (ssODN) template on the Cas ribonucleoprotein complex. The ssODN template is annealed to an extended guide RNA (gRNA) allowing its nuclear delivery at the right place, i.e. the targeted DNA cut, and at the right time. This new template import system is easy-to-design, easy-to-use, inexpensive and very versatile. It increases homology-directed repair (HDR) editing efficiency using Cas9 nuclease as demonstrated at many loci in many cell types (up to 12-fold higher HDR editing rate). Based on a heteroduplex gRNA-ssODN, it can also be used with the Cas9 nickase, resulting in HDR editing with minimal InDels, and preventing double-strand break (DSB)-mediated genotoxicity. ZE is a smart easy-to-use non-viral platform adaptable to targeted DSB (high HDR editing efficiency) or nick (high safety) to precisely model and correct a wide range of edits. It is suitable to many biological applications and could be considered for HDR-based gene therapies.

Lipid droplets (LDs) are neutral lipid storage organelles that emerge from the endoplasmic reticulum (ER). Their assembly occurs in ER regions enriched with seipin which, through its homooligomeric ring-like structure, facilitates neutral lipid nucleation. In yeast, seipin (Sei1) partners with Ldb16, Ldo45 (yeast homologue of human LDAF1) and Ldo16, which regulate LD formation and consumption. How the molecular architecture of the yeast seipin complex and its interaction with regulatory proteins adapt to different metabolic conditions remains poorly understood. Here, we show that multiple Ldb16 regions contribute differently to recruiting Ldo45 and Ldo16 to the seipin complex. Using an in-vivo site-specific photo-crosslinking approach, we further show that Ldo45 resides at the center of the seipin ring both in the absence and presence of neutral lipids. Interestingly, neutral lipid synthesis leads to the recruitment of Ldo45 but not Ldo16 to the complex. Our findings suggest that the seipin complex serves as a pre-assembled scaffold for lipid storage that can be remodeled in response to increased neutral lipid availability.

Macrophages infiltrate all human tissues where they play key roles in innate immunity, homeostasis, and tissue function. However, extensive clinical and experimental evidence indicates that macrophages also contribute significantly to the progression of several diseases such as cancer, cardiometabolic disorders, and inflammatory and neurodegenerative conditions. Advances in single-cell omics have revealed diverse macrophage populations in both healthy and diseased tissues. However, studying their functions is challenging due to limitations in tools for targeting specific populations. The Cre-lox system, involving Cre recombinase expression driven by macrophage-specific promoters, is widely used for gene manipulation. Despite its utility, this method has drawbacks like leaky expression, variable efficiency, and potential toxicity. Moreover, genetic models are costly and can have unintended effects on immune cells, hindering comprehensive studies on macrophage function. To address this challenge, we developed an advanced lipid nanoparticle-based system for precise RNA therapeutic delivery to macrophages, either broadly or via antibody-mediated targeting of specific subsets. This versatile platform enables the administration of various RNA molecules, such as mRNA, siRNA, and sgRNA for CRISPR/Cas9 applications, in both in vitro and in vivo settings. It allows for targeted cell depletion or gene knockout, facilitating detailed functional analysis. Furthermore, the system’s flexibility and precision are enhanced by its compatibility with Cre-specific Cas9 expression, enabling comprehensive genomic and proteomic targeting of specific macrophage subsets.

Acute lysosome damage triggers the endolysosome damage response (ELDR) in order to co-ordinate vesicle repair or removal by autophagy (lysophagy). However, it is unclear whether persistent damage, as occurs after chronic challenge to lysosome integrity, triggers wider cellular responses. Here, we show that longitudinal treatment with a lysosomotropic cancer therapeutic, the CDK4/6 inhibitor Palbociclib, invokes chronic lysosome damage in breast and lung cancer cells. Autophagy ameliorates but does not avert this phenotype, which persists over days. Damaged lysosomes form contacts with mitochondria, invoking mitochondrial stress and cytosolic efflux of immunostimulatory mitochondrial nucleic acids. Importantly, mitochondrial nucleic acid release is necessary for the anti-cancer interferon response to Palbociclib. In conclusion, chronic lysosome damage rewires cellular signalling responses through the mitochondrion and this effect should be considered when assessing the cellular actions of cancer therapeutics.

Cilia are highly conserved cellular organelles extruding from the surface of cell types carrying either sensory (signaling) or motile functions. These include photoreceptor cells and airway epithelia, where they function in light sensation and mucociliary clearance respectively. Retinitis pigmentosa GTPase regulator ( RPGR ) variants affect both photoreceptor sensory cilia and airway motile cilia, leading to retinitis pigmentosa (RP) and in some cases, primary ciliary dyskinesia (PCD), both debilitating conditions. Not all patients develop PCD and it is unclear which RPGR variants predispose patients to PCD and why this happens. In this study, using nasal biopsy samples of patients with RPGR -related RP, we leverage 2D organoid cell culturing, super-resolution microscopy, and live cell imaging to characterize the multiciliated cells from patients with different RPGR variants, healthy human nasal and bronchial multiciliated cells with CRISPR-modified RPGR function. We demonstrate for the first time that multiciliated cells with RPGR variants may have reduced ciliation, shorter cilia, significantly impaired cilia beat, or cilia beat incoordination, which could lead to defective mucociliary clearance and lung disease. In addition, we show the regulation of motile cilia by RPGR involves F-actin, as evidenced by temporarily reduced Gelsolin and undissolved condensed actin meshwork at the apical surface of RPGR-deficient multiciliated cells. In support, we show that the motile cilia defect can be ameliorated by treating with the actin polymerization inhibitor Latrunculin A. Though PCD was observed only in patients with variants that affect both main isoforms ( RPGR 1-19 and RPGR ORF15 ), patients with variants affecting only RPGR ORF15 also showed cilia and airway anomalies. Though all RPGR variants affected motile cilia in one way or another, RPGR loss of function variants affecting both isoforms are associated with more severe cilia and systemic phenotypes, the mechanisms of which involve the accumulation of apical F-actin. One Sentence Summary: Loss of RPGR, through the mechanism of apical F-actin accumulation in airway multiciliated cells, leads to reduced ciliation with short cilia that have an impaired beat, leading to defective mucociliary clearance.

After administering genome editors, their efficiency is limited by a multi-step process involving cellular uptake, trafficking, and nuclear import of the vector and its payload. These processes vary widely across cell types and differ depending on the nature and structure of the vector, whether it is a lipid nanoparticle or a different synthetic material. We developed a novel genome-wide CRISPR screening strategy to better understand these limitations within human cells to identify genes modulating cellular uptake, payload delivery, and gene editing efficiency. Our screen interrogates the cellular processes controlling genome editing by Cas-based nuclease and base editing strategies in human cells. We designed a genome-wide screen targeting 19,114 genes in HEK293 cells, and we identified six genes whose knockout increased nonviral editing efficiency in human cells by up to five-fold. Further validation through arrayed knockouts of the top hits from our screen boosted the editing efficiency from 5% to 50% when Cas9 was delivered via lipid-based nanoparticles. By designing the guides to target the screen library cassette, we could accurately track the library sgRNA identity and the editing outcome on the same amplicon via short-read sequencing, enabling the identification of rare outcomes via ‘computationally’ sorting edited from unedited cells within a heterogenous pool of >200M cells. In patient-derived human retinal pigment epithelium cells derived from pluripotent stem cells, BET1L, GJB2, and MS4A13 gene knockouts increased targeted genome editing by over five-fold. We anticipate that this high-throughput screening approach will facilitate the systematic engineering of novel nonviral genome editing delivery methods, where the identified novel gene hits can be further used to increase editing efficiency for other therapeutically relevant cell types.