Epithelial cancers such as stomach and ovary cancer tend to metastasize to the peritoneum, often leading to intractable disease and poor survival. Currently, the mechanisms that enable gastric cancer cells to penetrate the mesothelium, and to implant, invade, and survive in the peritoneal niche are poorly understood. To investigate these mechanisms, we developed a novel human peritoneal explant model. Briefly, fresh peritoneal tissue samples from abdominal surgery patients were cultured on top of a layer of GFP-labeled human gastric adenocarcinoma cells (AGS); 2% of these cells implanted into the peritoneum. The transcriptomic profile of the implanted AGS cells was compared to the profile of AGS cells that failed to implant using RNA sequencing. Differentially expressed genes were enriched significantly (fold change>2) for genes that enable cell adhesion, motility, and membrane depolarization. We compared this list of genes with a previously identified peritoneal metastasis whole exome sequencing dataset (SRP043661). Upon further analysis based on subcellular localization, cell adhesion and cytoskeletal organization, we found nine core “peritoneal implantation” genes. We functionally validated these genes with CRISPR knockout and assessed peritoneal implantation and invasion using the human peritoneal explant model described above. From these data we identified ADAM12 as a key player of peritoneal metastasis. Knock out of ADAM12 significantly impaired peritoneal metastasis in vivo and ex vivo . Exploration of three publicly available independent datasets indicated that ADAM12 is indeed clinically relevant in peritoneal metastasis. To explore the role of ITGAβ1 in mediating cell-matrix interactions in the presence of ADAM12, we performed ITGAβ1 pull-down assays followed by mass spectrometry analysis in ADAM12 WT cells. ADAM12 KO cells show a marked disruption of the ITGAβ1 interactome in GCa cells. Key cytoskeletal proteins such as MYH14, MYH10, MYH9, ACTA2, SPTN1 and TPM1–TPM4 were found to be interactors with ITGAβ1 in ADAM12 WT cells but not ADAM12 KO cells. Our approach and the new data identify a distinct peritoneal metastasis gene set that facilitates implantation and invasion of gastric cancer cells within the peritoneum. Disruption of these pathways with peritoneal-directed therapies has the potential to improve survival in patients with high-risk primary gastric cancer.

Summary Cancer immunotherapy is only effective in a subset of patients, highlighting the need for effective biomarkers and combination therapies. Here we systematically identify genetic determinants of cancer cell sensitivity to anti-tumor immunity by performing whole-genome CRISPR/Cas9 knock-out screens in autologous tumoroid-T cell co-cultures, isogenic cancer cell models deficient in interferon signaling, and in the context of four cytokines. We discover that loss of CHD1 and MAP3K7 potentiates the transcriptional response to IFN-γ, thereby creating an acquired vulnerability through sensitizing cancer cells to tumor-reactive T cells. Immune checkpoint blockade was more effective in a syngeneic mouse model of melanoma deficient in Chd1 and Map3k7 and was associated with elevated intra-tumoral CD8 + T cell numbers and activation. CHD1 and MAP3K7 are recurrently mutated in cancer and reduced expression in tumors correlates with response to immune checkpoint inhibitors in patients, nominating these genes as potential biomarkers of immunotherapy response.

ABSTRACT The development of traditional protein-targeted cancer therapies is a slow and arduous process, often taking years or even decades. In contrast, RNA-based therapies targeting crucial microRNAs (miRNAs) offer a faster alternative due to the sequence specific nature of miRNA inhibitor binding. This, combined with the capacity of individual miRNAs to influence multiple cellular pathways, makes these small RNAs attractive targets for cancer therapy. While miRNA are known to be dysregulated in prostate cancer (PCa), identifying their individual contributions to disease progression and the identification of therapeutically actionable miRNA targets in PCa has been challenging due to limited screening tools. To overcome this, we developed miRKOv2, a miRNA-only CRISPR knockout library enabling systematic, genome-wide loss-of-function screens to identify miRNAs essential for PCa cell survival. Our screens uncovered 69 potential essential miRNA candidates, with miR-483 demonstrating the most significant impact on PCa cell viability. Functional characterization demonstrated that miR-483 disruption significantly potentiated apoptosis in PCa cell lines. Mechanistically, we uncovered a novel regulatory axis wherein miR-483-3p directly modulates a BCLAF1/PUMA/BAK1 apoptotic signaling network, highlighting its critical role in maintaining PCa cell survival. Our findings provide novel insights into the complex regulatory role of miRNA in PCa progression and offer a potential therapeutic strategy for targeting miRNA-mediated pathways in metastatic disease.

DELLA proteins, members of the GRAS-domain family of transcriptional regulators, are critical for plant growth and development. They modulate transcription indirectly via interactions with hundreds of transcription factors. The phytohormone gibberellin (GA) triggers DELLA degradation, providing a mechanism by which plants can integrate developmental and environmental signals to regulate gene expression and optimize growth responses. In agriculture, DELLA mutations have been instrumental in improving crop performance. Most modern wheat ( Triticum aestivum L.) varieties carry Rht-B1b or Rht-D1b alleles that encode DELLA proteins resistant to GA-mediated degradation, resulting in constitutive partial suppression of stem growth, a semi-dwarf stature and lodging resistance. However, these alleles also reduce nitrogen use efficiency and early vigour, limiting their utility in some environments. Understanding how DELLA proteins regulate growth and development is, therefore, critical for refining breeding strategies. In this study, we identified the orthologous C2H2 zinc-finger transcription factors INDETERMINATE DOMAIN 5 ( IDD5 ) in wheat and SEMI-DWARF 3 ( SDW3 ) in barley ( Hordeum vulgare ) as positive regulators of stem and leaf expansion. Both IDD5 and SDW3 physically interact with, and act downstream of, DELLA proteins as key components of GA-mediated growth responses. Altered expression levels of GA biosynthesis genes suggest that IDD5 helps maintain GA homeostasis in addition to growth regulation. Loss-of-function mutations in IDD5 and SDW3 confer a GA-insensitive semi-dwarf phenotype comparable to that of the Rht-D1b ‘Green Revolution’ allele, highlighting their potential as novel dwarfing alleles for cereal improvement. Significance statement Our study identifies homologous wheat and barley transcription factors (IDD5 and SDW3) that interact with DELLA proteins to regulate plant height. Unlike conventional ‘Green Revolution’ DELLA mutations, which can reduce height but also have drawbacks such as lower nitrogen-use efficiency, these IDD genes may provide more targeted approaches to manage plant growth. By showing how IDD proteins promote stem and leaf expansion and by revealing their potential as alternative dwarfing alleles, our research opens new avenues both for fundamental research into plant growth pathways and for applications in cereal breeding. Ultimately, it could help produce crops with improved lodging resistance and fewer negative side effects than current dwarfing alleles.

Pest management has entered a new era with the emergence of three innovative antisense technologies: RNAi, CUAD, and CRISPR/Cas. These technologies, which operate through sequence-specific nucleic acid duplex formation and guided nuclease activity, offer unprecedented potential for targeted pest control. While RNA-guided systems such as RNAi and CRISPR/Cas were initially discovered in non-insect models as fundamental biological mechanisms (primarily in antiviral defense), the DNA-guided CUAD system was first identified in insect pests as a practical tool for pest control, while its broader role in ribosomal RNA (rRNA) biogenesis only recently recognized. These surprising discoveries have unveiled an entirely new dimension of gene regulation, with profound implications for sustainable pest management. Despite certain similarities of these technologies, RNAi, CUAD, and CRISPR/Cas differ in their mode of action, specificity, and applicability. No single approach provides a universal solution for all insect pests; instead, each is likely to be most effective against specific pest groups. Moreover, these technologies enable the rapid adaptation of pest management strategies by countering target-site resistance, ensuring long-term efficacy. This review provides a critical synthesis of the unique advantages and limitations of each antisense technology, highlighting their complementary roles in eco-friendly, nucleic acid-guided insect pest control. By bridging fundamental discoveries with applied research, we offer new perspectives on their practical implementation, underscoring the urgent need for their integration into modern pest management strategies.

The phospholipid scramblases Xkr8 and TMEM16F externalize phosphatidylserine (PS) by distinct mechanisms. Xkr8, is activated by caspase-mediated proteolytic cleavage, and in synergy with inactivation of P4-ATPase flippases, results in the irreversible externalization of PS on apoptotic cells and an “eat-me” signal for efferocytosis. In contrast, TMEM16F is a calcium activated scramblase that reversibly externalizes PS on viable cells via the transient increase in intracellular calcium in live cells. The tumor microenvironment (TME) is abundant with exposed PS, resulting from prolonged oncogenic and metabolic stresses and high apoptotic indexes of tumors. Such chronic PS externalization in the TME has been linked to host immune evasion from interactions of PS with inhibitory PS receptors such as TAM and TIM receptors. Here, in an effort to better understand the contributions of apoptotic vs live cell PS-externalization to tumorigenesis and immune evasion, we employed an E0771 orthotopic breast cancer model and genetically ablated Xkr8 and TMEM16F using CRISPR/Cas9. While neither the knockout of Xkr8 nor TMEM16F showed defects in cell intrinsic properties related to proliferation, tumor-sphere formation, and growth factor signaling, both knockouts suppressed tumorigenicity in immune-competent mice, but not in NOD/SCID or RAG-KO immune-deficient strains. Mechanistically, Xkr8-KO tumors suppressed macrophage-mediated efferocytosis, and TMEM16F-KO suppressed ER stress/calcium-induced PS externalization. Our data support an emerging idea in immune-oncology that constitutive PS externalization, mediated by scramblase dysregulation on tumor cells, supports immune evasion in the tumor microenvironment. This links apoptosis/efferocytosis and oncogenic stress involving calcium dysregulation, contributing to PS-mediated immune escape and cancer progression.

Extrachromosomal DNA (ecDNA) is a common source of oncogene amplification across many types of cancer. The non-Mendelian inheritance of ecDNA contributes to heterogeneous tumour genomes that rapidly evolve to resist treatment. Here, using single-cell and live-cell imaging, single-micronucleus sequencing, and computational modelling, we demonstrate that elevated levels of ecDNA predisposes cells to micronucleation. Damage on ecDNA, commonly arising from replication stress, detaches ecDNA from the chromosomes upon which they hitchhike during cell division, thereby causing micronucleus formation in daughter cells. Clusters of oncogene-containing, CIP2A-TOPBP1-associated ecDNA molecules form, and asymmetrically segregate into daughter cell micronuclei during cell division. ecDNA chromatin remains highly active during mitosis, but upon micronucleation, it undergoes suppressive chromatin remodeling, largely ceasing oncogene transcription. These studies provide insight into the fate of damaged ecDNA during cell division.

Summary The function of HSFs, known otherwise as ‘master thermoregulators,’ in plant developmental remains largely uninvestigated. In this study, we strategically analyze SlHSFB3a , a class B sub member of HSF transcription factor family, uniquely expresses in age-dependent tomato roots and improves root architecture by synchronizing auxin homeostasis. This data demonstrates SlHSFB3a overexpressed transgenics display higher lateral root (LR) density and early LR emergence improving root architecture. Generation of CRISPR-Knockout mutants displayed contrasting phenotype, confirming SlHSFB3a ’s vital role in root growth. In SlHSFB3a manipulated roots, concentration gradient auxin responses oscillated with increase in LR number. We highlight the signal transduction of SlHSFB3a mediated auxin activation that enhances tomato LRs. SlHSFB3a directly inhibits auxin repressors, increases auxin flow via ARF7/LOB20 pathway and positively modulates LR growth.

Abstract Intracellular parasites like Toxoplasma gondii scavenge host nutrients, particularly lipids, to support their growth and survival. Although Toxoplasma is known to adjust its metabolism based on nutrient availability, the mechanisms that mediate lipid sensing and metabolic adaptation remain poorly understood. Here, we performed a genome-wide CRISPR screen under lipid-rich (10% Fetal Bovine Serum (FBS)) and lipid-limited (1% FBS) conditions to identify genes critical for lipid-responsive fitness. We identified the Toxoplasma protein GRA38 as a lipid-dependent regulator of parasite fitness. GRA38 exhibits phosphatidic acid (PA) phosphatase (PAP) activity in vitro, which is significantly reduced by mutation of its conserved DxDxT/V catalytic motif. Disruption of GRA38 led to the accumulation of PA species and widespread alterations in lipid composition, consistent with impaired PAP activity. These lipid imbalances correlated with reduced parasite virulence in mice. Our findings identify GRA38 as a metabolic regulator important for maintaining lipid homeostasis and pathogenesis in Toxoplasma gondii. 

Abstract Human papillomavirus (HPV) infection is a major threat to women’s health worldwide. High-risk subtypes, particularly HPV16, require rigorous screening and long-term surveillance to control cervical cancer. However, traditional HPV testing is hampered by the need for nucleic acid extraction, reliance on specialized technicians, and fluorescence detection equipment, limiting its suitability for rapid on-site testing. In this study, we developed a Concanavalin A-assisted extraction-free one-pot recombinase polymerase amplification (RPA) CRISPR/Cas12a assay (ConRCA) for HPV16. Concanavalin A-coated magnetic beads were used for target enrichment and nucleic-acid-extraction-free processing. Suboptimal protospacer-adjacent motifs were used to achieve a one-pot RPA–CRISPR/Cas12a assay. The ConRCA assay can be completed in approximately 25 min under isothermal conditions and can detect at least 1.2 copies/µL of HPV16 genomic DNA using a fluorescence reader or test strip. The feasibility of this detection method was evaluated with 31 unextracted clinical samples. Compared with qPCR, the overall sensitivity was 95% (19/20), and the specificity was 100% (11/11). Our results indicate that the ConRCA assay has great potential utility as a point-of-care testing for the rapid identification of HPV.

Abstract Mild cognitive impairment (MCI) is an intermediate stage between normal cognition and dementia, with a high risk of progression to Alzheimer’s disease (AD). As an important threshold for the intervention and prevention of AD, accurate diagnosis of AD-related MCI based on plasma biomarkers is crucial. However, early detection of AD-related MCI still poses a huge challenge. Herein, we propose an ultrasensitive CRISPR-based multi-protein detection array (UCMDA) that integrates the array device with CRISPR/Cas12a and antibody pair-based multi-recombinase polymerase amplification to facilitate inexpensive, sensitive, and specific detection of multiple markers in plasma. With only one fluorescence probe, UCMDA can simultaneously detect Aβ40, Aβ42, p-tau181, p-tau217, p-tau231, and p-tau396,404 within 1 h with a detection limit of 1 fg/mL. The applicability of UCMDA was demonstrated in 155 clinical plasma samples from normal senile controls (NCs), individuals with AD-related MCI, and patients with AD. We found that the UCMDA platform combined with machine learning algorithms achieved accurate early diagnosis of AD-related MCI with a detection accuracy, sensitivity, and specificity exceeding 90.38%. We anticipate that this inexpensive, ultrasensitive, and multi-detection platform can also be widely used to detect other protein-related biomarkers besides AD.

While prime editing offers improved precision compared to traditional CRISPR-Cas9 systems, concerns remain regarding potential off-target effects, including epigenetic changes such as DNA methylation. In this study, we investigated whether prime editing induces aberrant CpG methylation patterns. Whole-genome bisulfite sequencing revealed overall methylation similarity between Cas9-edited, and PE2-edited cells. However, localized epigenetic changes were observed, particularly in CpG islands and exon regions. The PE2-edited group showed a higher proportion of differentially methylated regions (DMRs) in some coding sequences compared to controls and Cas9-edited samples. Notably, CpG island methylation reached 0.18% in the PE2 vs. Cas9 comparison, indicating a higher susceptibility of these regulatory elements to epigenetic alterations by prime editing. Gene ontology and KEGG pathway analyses further revealed enrichment in molecular functions related to transcriptional regulation and redox activity in PE2-edited cells. These findings suggest that prime editing, while precise, may introduce subtle but functionally relevant methylation changes that could influence gene expression and cellular pathways. In summary, prime editing can induce localized DNA methylation changes in human cells, particularly within regulatory and coding regions. Understanding these epigenetic consequences is critical for the development of safer and more effective therapeutic applications of genome editing technologies.

Sirex noctilio is an invasive pest of pine that has caused significant economic damage in South Africa and many other Southern Hemisphere countries. Current management tools are not efficient in all cases and consequently there is a need for more efficient and targeted control measures. An emerging tool for pest management is the use of gene editing and associated gene drive systems. In this study, we aim to investigate the use of CRISPR-Cas gene drive systems in the management of S. noctilio in South Africa. As a first step, we developed a model for the population dynamics of S. noctilio , using historical national population monitoring data and incorporating the influence of two main biological control agents of the pest. We then modelled the influence of two different CRISPR-Cas systems on the population dynamics of S. noctilio namely, a baseline CRISPR model and Complementary Sex Determination CRISPR (CSD) model. Each model is used to simulate a male and female only introduction strategy to estimate the effectiveness of different methods of introducing the gene drive system. The model calibration was achieved by optimizing the model fit to existing data using the least squares technique. Results suggest that both CRISPR gene drive systems would be effective at controlling the population growth of S. noctilio at high levels of introduction, but overall population control would be hindered by practical limitations. Although only two CRISPR models were explored, the underlying population model serves as a framework for further studies into the population dynamics of Sirex noctilio , as well as many other CRISPR-Cas gene drive systems.

ABSTRACT Objectives The defense mechanisms in bacterial pathogens protect them from host immune systems, bacteriophage infections and stringent environmental conditions. This study explores the defense-systems in multidrug resistant Klebsiella pneumoniae isolated from Ghanaian hospital ICUs focusing on CRISPR-cas, restriction-modification and toxin-antitoxin systems (TAs). Method Genomic DNA of K. pneumoniae environmental (NS2) and clinical (PS4) strains were subjected to whole genome sequencing using Illumina and assembled with SPAdes (v3.13.1). CRISPR-cas, restriction-modification and TAs were identified using PADLOC, defense finder and TADB3.0 respectively. Results The strains harbor diverse defense systems. Relative to reference K. pneumoniae with 10 defense systems, NS2 has twelve and PS4, five. CRISPR-Cas systems were found only in NS2, while both strains have type I, II and IV restriction and modification systems. The strains have > 30 characterized and novel TAs (type I, II, IV, VIII) similar to reference K. pneumoniae . NS2 harbors more TAs than PS4 both on chromosomes and plasmids. The strains have comparable resistance determinants to more than six classes of antibiotics. Conclusion The genome of strains encodes similar clinically relevant defense-systems indicating possibility of microbial exchange from fomites and humans. They could leverage the defense-systems to propagate resistance in high-risk environments such as the hospital. Fomite-resident strain with high levels of resistance could increase infection risk in the ICU; hence, should also be prioritized.

Some RNA viruses package their genomes with extraordinary selectivity, assembling protein capsids around their own viral RNA while excluding nearly all host RNA. How the assembling proteins distinguish viral RNA from host RNA is not fully understood, but RNA structure is thought to play a key role. To test this idea, we perform in-cellulo packaging experiments using bacteriophage MS2 coat proteins and a variety of RNA molecules in E. coli . In each experiment, plasmid-derived RNA molecules with a specified sequence compete against the cellular transcriptome for packaging by plasmid-derived coat proteins. Following this competition, we quantify the total amount and relative composition of the packaged RNA using electron microscopy, interferometric scattering microscopy, and high-throughput sequencing. By systematically varying the input RNA sequence and measuring changes in packaging outcomes, we are able to directly test competing models of selective packaging. Our results rule out a longstanding model in which selective packaging requires the well-known TR stem-loop, and instead support more recent models in which selectivity emerges from the collective interactions of multiple coat proteins and multiple stem-loops distributed across the RNA molecule. These findings establish a framework for understanding selective packaging in a range of natural viruses and virus-like particles, and lay the groundwork for engineering synthetic systems that package specific RNA cargoes. Significance Statement Bacteriophage MS2 packages its RNA genome into protective protein shells called capsids while excluding nearly all host-cell RNA. Engineering synthetic capsids with similar selectivity could enable a broad range of RNA-based technologies, including CRISPR gene editing systems, mRNA vaccines, and other emerging RNA-based therapeutics. Our study shows that selective packaging in MS2 is not dictated by a single, high-affinity RNA-protein interaction but instead emerges from the collective interactions of multiple coat proteins and an ensemble of stem-loops distributed across the RNA molecule. By establishing these collective interactions as the basis of selectivity, our findings provide a foundation for engineering synthetic capsids capable of selectively packaging target RNAs for next-generation RNA-based technologies.

Long noncoding RNAs (lncRNAs), non-protein-coding transcripts exceeding 200 nucleotides, are critical regulators of gene expression through chromatin remodeling, transcriptional modulation, and post-transcriptional modifications. While ionizing radiation (IR) induces cellular damage through direct DNA breaks, reactive oxygen species (ROS)-mediated oxidative stress, and bystander effects, the functional involvement of lncRNAs in radiation response remains incompletely characterized. Here, through genome-wide CRISPR activation (CRISPRa) screening in non-small cell lung cancer (NSCLC) cells, we identified LOC401312 as a novel radiosensitizing lncRNA, the stable overexpression of which significantly enhanced IR sensitivity. Transcriptomic profiling revealed that LOC401312 transcriptionally upregulates carbamoyl-phosphate synthase 1 (CPS1), a mitochondrial enzyme involved in pyrimidine biosynthesis. Notably, CPS1 overexpression recapitulated the radiosensitization phenotype observed with LOC401312 activation. Mechanistic investigations revealed that CPS1 suppresses the phosphorylation of ATM kinase (Ser1981) and XRCC1 protein levels, which are key mediators of DNA damage checkpoint activation and base excision repair, respectively. This study establishes the LOC401312-CPS1-ATM/XRCC1 axis as a previously unrecognized regulatory network governing radiation sensitivity, highlighting the potential of lncRNA-directed metabolic rewiring to impair DNA repair fidelity. Our findings not only expand the functional landscape of lncRNAs in DNA damage response but also provide a therapeutic rationale for targeting the LOC401312-CPS1 axis to improve radiotherapy efficacy in NSCLC.

The primate brain possesses unique physiological and developmental features whose systematic investigation is hampered by a paucity of transgenic germline models and tools. Here, we present a minimally invasive method to introduce transgenes widely across the primate cerebral cortex using ultrasound-guided fetal intracerebroventricular viral injections (FIVI). This technique enables rapid-onset and long-lasting transgene expression following the delivery of recombinant adeno-associated viruses (rAAVs). By adjusting the gestational timing of injections, viral serotypes, and transcriptional regulatory elements, rAAV FIVI allows for systematic targeting of specific cell populations. We demonstrate the versatility of this method through restricted laminar expression in the cortex, Cre-dependent targeting of neurons, CRISPR-based gene editing, and labeling of peripheral somatosensory and retinal pathways. By mimicking key desirable features of germline transgenic models, this efficient and targeted method for gene transfer into the fetal primate brain opens new avenues for experimental and translational neuroscience across the lifespan.

Abstract Epithelial ovarian cancer (EOC) metastasizes predominantly through multicellular aggregates known as spheroids, which disseminate within the peritoneal cavity and initiate secondary disease upon reattachment at distant sites. EOC spheroids resist detachment-induced cell death by upregulating stress responses including AMP-activated protein kinase (AMPK) signaling and AMPK-dependent macroautophagy (autophagy), highlighting these pathways as potential therapeutic targets. Previously, we used a pharmacological approach to putatively identify Ca 2+ /calmodulin-dependent protein kinase kinase 2 (CAMKKβ, encoded by CAMKK2 ) as the primary activator of AMPK in EOC spheroids. Herein we have generated CAMKK2 knockout EOC cell lines via CRISPR–Cas9 genome editing to confirm this function of CAMKKβ and explore the impacts of its loss using in vitro and in vivo models of metastatic EOC. CAMKK2 knockout spheroids exhibited decreased AMPK activation, autophagic flux, cell viability, and metastatic potential relative to parental spheroids, and intraperitoneal xenograft tumours lacking CAMKKβ grew slower than their CAMKKβ-intact counterparts. Effect magnitudes varied between cell line models, suggesting context-dependent roles for CAMKKβ in EOC and rationalizing further studies to characterize the underlying mechanisms. Altogether, our findings highlight CAMKKβ as an important contributor to metabolic reprogramming in EOC spheroids and as a potential therapeutic target in the setting of advanced disease.

Genome editing has the potential to treat genetic disorders at the source. This can be achieved by modifying the defective DNA through the intentional insertion, deletion, or substitution of genomic content. Among all genome editing technologies, CRISPR/Cas (Clustered Regularly Interspaced Short Palindromic Repeats and CRISPR-associated protein) is considered the gold standard. CRISPR/Cas uses a single guide RNA (sgRNA) to direct the Cas nuclease to a target DNA region. Due to the ease at creating small RNA molecules, it is possible to have the CRISPR/Cas complex target any arbitrary DNA sequence, thus making it a versatile tool. The efficacy of the complex is dependent on the ability of the sgRNA to bind to a complementary DNA sequence, which varies based on the sequence. Thus, a major challenge is finding sgRNA sequences that have good efficacy. This is where computational models can aid scientists: by predicting the activity of sgRNAs to help narrow the search space of finding the optimal sgRNA. We have used a large new dataset to build and compare the ability of several different machine learning architectures’ ability to predict on-target CRISPR/Cas activity. Additionally, we explored how adding GC content affects our sgRNA activity predictions. Our novel hybrid model, ChromeCRISPR, combines the strengths of Convolutional Neural Networks (CNN) with Recurrent Neural Network (RNN) models, has outperformed state-of-the-art models, including DeepHF and AttCrispr, establishing a new benchmark for predictive accuracy in CRISPR/Cas9 efficacy predictions.

RECQL4 encodes a RecQ helicase, one of a family of DNA unwinding enzymes with roles in DNA replication, double strand break repair and genomic stability. Pathogenic variants in RECQL4 are clinically associated with three rare autosomal recessive conditions: Rothmund-Thomson Syndrome type II, Baller-Gerold Syndrome and RAPADILINO syndrome. These three syndromes show overlapping growth retardation, low bone density and skeletal defects affecting the arms and hands. Here, we take advantage of the ability to generate one-sided CRISPR knockdowns of recql4 in Xenopus laevis tadpoles. Tadpoles develop normally until feeding starts, after which growth slows on the edited side leading to a curved posture, smaller eyes (micropthalmia) and reduced head size (microcephaly). Forelimb buds fail to develop, leading to complete absense of the forelimb on the edited side. Additionally, Meckel’s cartilage (lower jaw) ossification is absent or reduced and the hyoid cartilage is smaller, but this is not due to deficiencies in cranial neural crest migration on the edited side. Knockdown of recql4 also results in hypoplastic vasculature, with reduced branching from the aorta on the edited side. Taken together, our results clearly show the utility of unilateral CRISPR editing in Xenopus for understanding the specific phenotypic developmental effects of mutations affecting cell proliferation.

Abstract Regulated in development and DNA damage response-1 (REDD1/DDIT4) is induced in response to environmental stress to restrain the mechanistic target of rapamycin complex 1 (mTORC1) signaling as an adaptive strategy to restore cellular homeostasis. Interestingly, REDD1/DDIT4 expression is upregulated in several tumour types including colorectal cancer, suggesting it may have a role in tumourigenesis. Here, we report that activating transcription factor 4 (ATF4)-dependent REDD1/DDIT4 expression is required for survival of colon tumour cells undergoing endoplasmic reticulum (ER) stress through the modulation of TRAILR2/DR5 gene expression. Our findings further demonstrate that resistance to ER stress-induced apoptosis in multicellular tumour spheroids (MCTS) is associated with constitutive expression of REDD1/DDIT4 and diminished mTORC1 activity. CRISPR/Cas9-mediated deletion of REDD1/DDIT4 markedly increases TRAILR2/DR5 expression and enhances apoptosis in spheroids exposed to ER stress. Interestingly, RNA sequencing analysis reveals that the loss of the transcriptional regulator MECOM/EVI-1, a partner of the corepressor protein C-terminal Binding Protein (CtBP), in cells deficient in REDD1/DDIT4 amplifies the ER stress-induced upregulation of TRAILR2/DR5, leading to enhanced apoptosis. In summary, our findings underscore the crucial role of REDD1/DDIT4 in regulating TRAILR2/DR5-induced caspase-8 activation and apoptosis under chronic ER stress, by inhibiting mTORC1 activity and promoting MECOM/EVI-1-mediated suppression of TRAILR2/DR5 gene expression.

Abstract Epithelial ovarian cancer (EOC) is a leading cause of gynecological cancer mortality, driven largely by late diagnosis and chemo-resistant disease. While autophagy plays a critical role in the survival of EOC spheroids during metastasis, the role of ULK1, a key regulator of autophagy, in EOC progression remains unclear. To investigate this, we utilized CRISPR/Cas9 technology to delete ULK1 in EOC cell lines OVCAR8 and HEYA8, and the immortalized fallopian tube epithelial cell line FT190. Immunoblotting confirmed ULK1 loss and its associated autophagy disruption in EOC spheroids, evidenced by reduced Beclin-1 phosphorylation, impaired LC3 processing, and p62 accumulation. Culture-based assays revealed that ULK1 knockout decreased EOC spheroid cell viability due to increased apoptosis and, notably, impaired matrix-bound organoid growth, offering new insights into the potential role of ULK1 in affecting EOC tumor growth and spread. These findings were further demonstrated by in vivo xenograft models, in which ULK1 loss significantly reduced tumor burden and metastatic potential. The potential for ULK1 requirement in metastatic properties was supported by diminished invasive capacity of ULK1 knockout spheroid cells in mesothelial clearance assays. To investigate the mechanisms by which ULK1 contributes EOC tumor progression and metastasis, we conducted proteomic analyses of OVCAR8 spheroids, which revealed that ULK1 loss disrupted critical signaling pathways, including MEK-MAPK, PI3K-AKT-mTOR, and apoptosis regulation. Although ULK1 knockout failed to synergize with standard-of-care chemotherapeutics, it significantly enhanced sensitivity to MEK and mTOR inhibition, revealing potential therapeutic combinations to target autophagy via ULK1 and MAPK and PI3K-AKT-mTOR pathway vulnerabilities in EOC. Overall, this study highlights ULK1 as a critical regulator of multiple steps of EOC growth and metastasis, underscoring its potential as a novel therapeutic target in advanced ovarian cancer.

Abstract Autosomal recessive spastic ataxia of Charlevoix-Saguenay (ARSACS ) is an early-onset neurodevelopmental and neurodegenerative disorder characterized by ataxia, spasticity, and peripheral neuropathy. However, several studies have highlighted that some patients also experience cognitive, emotional and social deficits, suggesting a more complex clinical picture that extends beyond motor symptoms. Building on these findings, this study aimed to: i) investigate locomotor, social and cognitive deficits in adult sacs -/- zebrafish versus wild-type (WT) controls through behavioural tests; ii) identify molecular patterns associated with the adult disease phenotype using transcriptomic and proteomic analyses of sacs -/- and WT brains; iii) evaluate the effectiveness of long-term treatment with tauroursodeoxycholic acid (TUDCA) on behavioural outcomes and omics profiles in the zebrafish sacs -/- model. Our findings indicate impairments in cognitive, social, and emotional behaviors in aged sacs -/- zebrafish, which resemble some deficits observed in human patients. Transcriptomic and proteomic analyses of adult brains identified alterations in genes related to circadian rhythms and neuroinflammation. Notably, disruptions in sleep and circadian rhythms are frequently reported in individuals with cerebellar ataxia and may contribute to cognitive and behavioral changes. Long-term treatment with TUDCA, a neuroprotective molecule, was associated with partial improvements in social and cognitive behaviors and modifications in omics profiles in the zebrafish model. These results support the potential of further exploring TUDCA in future preclinical and clinical studies, while also emphasizing the need for additional investigations to better understand its mechanisms of action.

The degradation of aggregation-prone tau is regulated by the ubiquitin-proteasome system (UPS) and autophagy, which are impaired in Alzheimer’s disease (AD) and related tauopathies causing tau aggregation. Protein ubiquitination with linkage specificity determines the fate of proteins that can be either degradative or stabilization signals. While the linear M1-linked ubiquitination on protein aggregates is a signaling hub that recruits various ubiquitin-binding proteins for coordinated actions of protein aggregates turnover and inflammatory NF-kB activation, the deubiquitinase OTULIN counteracts with the M1-linked ubiquitin signaling. However, the exact role of OTULIN on tau aggregate clearance in AD is unknown. Based on our bulk RNA sequence analysis, human inducible pluripotent stem cell (iPSC)-derived neurons (iPSNs) from an individual with late-onset sporadic AD (sAD2.1) show downregulation of ubiquitin ligase activating factors (MAGEA2B and MAGEA) and OTULIN long non-coding RNA (lncRNA-OTULIN) compared to healthy control WTC11 iPSNs. In sAD2.1 iPSNs, downregulated lncRNA-OTULIN is inversely correlated with increased levels of OTULIN protein and phosphorylated tau at p-S202/p-T205 (AT8), p-T231 (AT180), and p-S396/p-S404 (PHF-1). Loss of OTULIN deubiquitinase function using pharmacological inhibitor UC495 or CRISPR-Cas9-mediated OTULIN gene knockout causes a significant reduction of total tau and phosphorylated tau at AT8 epitope in sAD2.1 iPSNs. Whereas in SH-SY5Y neuroblastoma cells, either treating with the UC495 compound or knocking out of the OTULIN gene causes a significant reduction of total tau at both mRNA and protein levels and consequently decreases phosphorylated tau at AT8, AT180, and PHF-1 epitopes. An additional bulk RNA sequence analysis of OUTLIN knockout SH-SY5Y shows a 14-fold down-regulation of tau mRNA levels and differential expression of many other genes associated with autophagy, UPS, NF-kB pathway, and RNA metabolism. Together, our results suggest for the first time a non-canonical function for OTULIN in regulating gene expression and RNA metabolism, which may have a significant pathogenic role in AD and related tauopathies.

The capacity to engineer organisms with multiple transgenic components is crucial to synthetic biology and basic biology research. For the former field, transgenic organisms allow the creation of novel biological functions; for the latter, such organisms provide potent means of dissecting complex biological pathways. However, the size limitations of a single transgenesis event and challenges associated with the assembly of multiple DNA fragments hinder the efficient integration of multiple transgenes. To overcome these hurdles, here we introduce a building block for synthetic design termed an integrated genetic array (IGA), which incorporates all genetic components into a single locus to prevent their separation during genetic manipulations. Since the natural recombination rate for genes located in the same locus is near zero, to construct IGAs we developed the Super Recombinator (SuRe) system, which uses CRISPR/Cas9, alone or in combination with site-specific serine recombinases, for in vivo transgene recombination at a single genomic locus. SuRe effectively doubles the number of elements assembled in each recombination round, exponentially accelerating IGA construction. By preventing the separation of transgenic elements, SuRe greatly reduces screening burdens, as validated here through studies of Drosophila melanogaster and Caenorhabditis elegans . To optimize SuRe, we compared CRISPR/Cas9-induced homology-directed recombination to site-specific recombination using various serine recombinases. Optimized versions of SuRe achieved efficiency and fidelity values near their theoretical maxima and allowed the generation of recombinant products up to 4.2 Mbp in size in Drosophila . Using SuRe, we created fruit flies with 12 transgenic elements for fluorescence voltage imaging of neural activity in precisely defined cell-types. Mathematical modeling of the scalability of SuRe to large transgene assemblies showed that integration times and gene assembly workloads respectively scale logarithmically and linearly with the number of transgenes, both major improvements over conventional approaches. Overall, SuRe enables the efficient integration of multiple genes at individual loci, up to the chromosomal scale.