κB-Ras/RalGAP complexes limit the activity of Ral GTPases, which function in EGFR/Ras signaling. RalGAP expression is down-regulated in pancreatic cancer; however, the role of RalGAP and Ral GTPases in tumor development in vivo remained unclear. Here, we show that pancreatic RalGAPβ deficiency alone is sufficient to induce inflammation and neoplasia in vivo. We identify that this phenotype is triggered by disturbance of the secretory pathway and polarized exocytosis in acinar cells, demonstrating that RalGAP complexes uphold spatial control of Ral activity. We furthermore show that RALGAPβ deficiency results in defective primary cilium assembly, a process required for efficient acinar regeneration upon inflammation. Only primary cilium formation depends on κB-Ras proteins, suggesting that κB-Ras proteins are not essential for all RalGAP complex-controlled processes. In combination with an oncogenic KRAS G12D mutation, RalGAPβ deficiency leads to a dramatic shortening of tumor latency and median survival. Our results highlight an important role of RalGAP/Ral signaling in upholding acinar cell identity and preventing pancreatic cancer development.

Multiple myeloma (MM) is a B-cell malignancy accounting for 20 % of all blood-associated cancers. MM patients with a poorer prognosis and high-risk stratification were previously observed to be causally linked to the constitutive activation of non-canonical NF-κB (ncNF-κB) pathway. Consistent with this, the ncNF-κB p52 transcription factor was earlier found to regulate the enhancer landscape of MM to potentiate oncogenic transcription. However, the mechanism by which aberrant p52 expression is involved in coordinating enhancer activity has not been well explored. In this study, we analysed H3K27ac ChIP-seq and ATAC-seq data from MM cell lines and patient samples to screen for putative transcription factors that cooperate with p52 to regulate enhancers activated in MM. We report that ATF4 interacts with p52 and together, this complex mediates the activity of a subset of MM-associated enhancers through the recruitment of histone acetyltransferases (HATs), p300 and CBP (CREB-binding protein). We also identified a ATF4:p52 regulated target gene BACH1 under the regulation of a proximal super-enhancer, which was found to drive oncogenesis in MM by promoting cell cycle progression and proliferation. Together, our findings provide further mechanistic insights into how aberrant enhancer activation observed in MM tumours could lead to disease progression.

Laboratory-based detection of D sites is laborious and expensive. In this study, we developed effective machine learning models employing efficient feature encoding methods to identify D sites. Initially, we explored various state-of-the-art feature encoding approaches and 30 machine learning techniques for each and selected the top eight models based on their independent testing and cross-validation outcomes. Finally, we developed DHUpredET using the extra tree classifier methods for predicting DHU sites. The DHUpredET model demonstrated balanced performance across all evaluation criteria, outperforming state-of-the-art models by 8 % and 14 % in terms of accuracy and sensitivity, respectively, on an independent test set. Further analysis revealed that the model achieved higher accuracy with position-specific two nucleotide (PS2) features, leading us to conclude that PS2 features are the best suited for the DHUpredET model. Therefore, our proposed model emerges as the most favorite choice for predicting D sites. In addition, we conducted an in-depth analysis of local features and identified a particularly significant attribute with a feature score of 0.035 for PS2_299 attributes. This tool holds immense promise as an advantageous instrument for accelerating the discovery of D modification sites, which contributes too many targeting therapeutic and understanding RNA structure.

Cancer is a complicated disease that results from the interplay between specific changes in cellular genetics and diverse microenvironments. The application of high-performance and customizable clustered regularly interspaced palindromic repeats/associated protein (CRISPR/Cas) nuclease systems has significantly enhanced genome editing for accurate cancer modeling and facilitated simultaneous genetic modification for cancer therapy and mutation identification. Achieving an effective CRISPR/Cas platform for cancer treatment depends on the identification, selection, and optimization of specific mutated genes in targeted cancer tissues. However, overcoming the off-target effects, specificity, and immunogenicity are additional challenges that must be addressed while developing a gene editing system for cancer therapy. From this perspective, we briefly covered the pipeline of CRISPR/Cas cancer therapy, identified target genes to optimize gRNAs and sgRNAs, and explored alternative delivery modalities, including viral, non-viral, and extracellular vesicles. In addition, the list of patents and current clinical trials related to this unique cancer therapy method is discussed. In summary, we have discussed comprehensive start-to-end pipeline strategies for CRISPR/Cas development to advance the precision, effectiveness, and safety of clinical applications for cancer therapy.

Cell lineage tracing is a key technology for describing the developmental history of individual progenitor cells and assembling them to form a lineage development tree. However, traditional methods have limitations of poor stability and insufficient resolution. As an efficient and flexible gene editing tool, CRISPR-Cas9 system has been widely used in biological research. Furthermore, CRISPR-Cas9 gene editing-based tracing methods can introduce fluorescent proteins, reporter genes, or DNA barcodes for high-throughput sequencing, enabling precise lineage analysis, significantly improving precision and resolution, and expanding its application range. In this review, we summarize applications of CRISPR-Cas9 system in cell lineage tracing, with special emphasis on its successful applications in traditional model animals (e.g., zebrafish and mice), large animal models (pigs), and human cells or organoids. We also discussed its potential prospects and challenges in xenotransplantation and regenerative medicine.

Effective therapies for cancers relying on the alternative lengthening of telomeres (ALT) mechanisms are still needed. Here, using CRISPR-Cas9 strategies, we validate FANCM (Fanconi anemia complementation group M) as a crucial target for ALT-associated cancers and demonstrate its importance in both in vitro and in vivo models. We further explore the use of antisense oligonucleotides (ASOs), specifically gapmers, to target FANCM mRNA. We designed and screened several gapmers, identifying effective candidates that potently reduced FANCM expression, which led to an increased ALT activity and telomeric dysfunction, concomitant with a reduced viability of ALT-positive cancer cells. Notably, gapmer 14, one of the identified ASOs, significantly impaired the viability of ALT cells and reduced tumor growth in an ALT-positive liposarcoma xenograft model, highlighting its therapeutic potential. These findings suggest that FANCM-targeting ASOs could represent a promising effective strategy for treating ALT-positive cancers.

Coordination of adaptive metabolism through signaling networks is essential for cellular bioenergetics and homeostasis. Phosphorylation of metabolic enzymes provides a rapid, efficient, and dynamic mechanism to regulate metabolic networks. Our structural analysis stratified phosphosites on metabolic enzymes based on proximity to functional and dimerization domains. Most phosphosites occur on oxidoreductases and are enriched near substrate, cofactor, active sites, or dimer interfaces. Despite low stoichiometry, phosphotyrosine (pY) is overrepresented in functional domains. Using high-fat diet (HFD)-induced obesity in C57BL/6J mice and multiomics, we measured HFD-induced sex-specific dysregulation of pY and metabolites, which was reversible with the antioxidant butylated hydroxyanisole (BHA). Computational modeling revealed predictive pY sites for HFD- or BHA-induced metabolite changes. We characterized functional roles for predictive pY sites on glutathione S-transferase pi 1 (GSTP1), isocitrate dehydrogenase 1 (IDH1), and uridine monophosphate synthase (UMPS) using CRISPR interference (CRISPRi) rescue and stable isotope tracing. Our findings reveal mechanisms whereby cellular signaling fine-tunes enzyme activity and metabolism.

Monocytes infiltrating tumors acquire various states that distinctly impact cancer treatment. Here, we show that resistance of tumors to radiotherapy (RT) is controlled by the accumulation of monocyte-derived dendritic cells (moDCs). These moDCs are characterized by the expression of CD301b and have a superior capacity to generate regulatory T cells (Tregs). Accordingly, moDC depletion limits Treg generation and improves the therapeutic outcome of RT. Mechanistically, we demonstrate that granulocyte-macrophage colony-stimulating factor (GM-CSF) derived from radioresistant tumor cells following RT is necessary for the accumulation of moDCs. Our results unravel the immunosuppressive function of moDCs and identify GM-CSF as an immunotherapeutic target during RT.

The increasing changes in the climate patterns across the globe have deeply affected food systems where unparalleled and unmatched challenges are created. This jeopardizes food security due to an ever-increasing population. The extreme efficiency of C4 crops as compared to C3 crops makes them incredibly significant in securing food safety. C4 crops, maize (Zea mays L.) and sorghum (Sorghum bicolor L. Moench) in particular, have the ability to withstand osmotic stress induced by oxidative stress. Osmotic stress causes a series of physical changes in a plant thus facilitating reduced water uptake and photosynthesis inhibition, such as membrane tension, cell wall stiffness, and turgor changes. There has been a great advancement in plant breeding brought by introduction of clustered regularly interspaced short palindromic repeats (CRISPR) gene editing technology. This technology offers precise alterations to an organism's DNA through targeting specific genes for desired traits in a wide number of crop species. Despite its immense opportunities in plant breeding, it faces limitations such as effective delivery systems, editing efficiency, regulatory concerns, and off-target effects. Future prospects lie in optimizing next-generation techniques, such as prime editing, and developing novel genotype-independent delivery methods. Overall, the transformative role of CRISPR/Cas9 in sorghum and maize breeding underscores the need for responsible and sustainable utilization to address global food security challenges.

Gene editing technology based on clustered regularly interspaced short palindromic repeats/associated protein (CRISPR/Cas) systems serves as an efficient tool in cancer therapy. Tracking the gene editing process can help identify the progress of cancer treatment. However, existing techniques for monitoring the gene editing process rely on lysed cells, which can not reflect the dynamic changes of nucleic acid in living cells. It urgently needs in situ and real-time imaging technologies to track the gene editing process at a living single-cell level more effectively and precisely. Here, we reported a highly efficient nuclear-targeted material, phenylboronic acid modified linear PEI (LPBA), for loading gene editing plasmids and fluorescent probes to track gene editing processes of microRNA. Based on LPBA, we achieved efficient intranuclear microRNA imaging at the living cell level, reaching 32.4-fold higher than the linear PEI (LPEI) delivery system, which facilitated further sensitive monitoring of the gene editing process both in living cells and in vivo. Meanwhile, this efficient gene-editing and real-time detection technique could be extended to screening effective gene-editing plasmids. Such LPBA-based imaging technology extended the imaging area of microRNA and offered new insight in the field of gene editing and nucleic acid detection.

IL-17-producing lymphocytes are involved in both tissue repair and the propagation of inflammation, with their effects highly context-dependent. Mucosal-Associated-Invariant-T-cells (MAIT), a subset of innate-like T cells with features of both Th1 and Th17 lineages, are increasingly recognized for their roles in mucosal immunity. Here, we identified the Collaborative-Cross CC011/Unc strain, which spontaneously develops chronic colitis, as being enriched for MAIT cells. This expansion coincides with an age-related loss of intestinal barrier permeability and colonic inflammation. Microbiota from CC011 mice activated MAIT cells in an MR1-dependent manner and selectively promoted the accumulation of MAIT17 cells in peripheral tissues. Single-cell transcriptomic analyses revealed colon MAIT cells from colitic CC011 mice expressed a pathogenic Th17-like signature, characterized by IL-1 and IL-23 signaling, IL-17A and IFNγ co-expression, and upregulation of IL-23R, features that correlated with inflammatory Ly6Chi monocyte abundance. Genetic deletion of Traj33, essential for MAIT development, significantly reduced colonic inflammation in this model. These findings demonstrate that MAIT cells integrate microbial and cytokine cues to adopt a pathogenic effector phenotype that exacerbates chronic intestinal inflammation.

Stem cell-based embryo models (SEMs) have the potential to transform our understanding of early human embryogenesis. A critical step in engineering SEMs is the generation of the major cell types that compose preimplantation embryos including two primary extraembryonic lineages: (i) trophoblast cells, which are crucial for implantation and the establishment of maternal-fetal exchange, and (ii) hypoblast cells, which contribute to yolk sac formation. In addition, both cell types provide key signaling cues necessary for embryonic development. CRISPR-based epigenome editors are programmable devices that allow for efficient and precise activation (CRISPRa) or repression (CRISPRi) of cell fate-determining factors by modulating endogenous regulatory elements. Here, we present a step-by-step method to implement CRISPRa for controlling cell fate in embryonic stem cells based on our work in generation of CRISPR-programmed mouse embryo models.

The ability to robustly predict guide RNA (gRNA) activity is a long-standing goal for CRISPR applications, as it would reduce the need to pre-screen gRNAs. Quantification of formation of short insertions and deletions (indels) after DNA cleavage by transcribed gRNAs has been typically used to measure and predict gRNA activity. We evaluate the effect of chemically synthesized Cas9 gRNAs on different cellular DNA cleavage outcomes and find that the activity of different gRNAs is largely similar and often underestimated when only indels are scored. We provide a simple linear model that reliably predicts synthetic gRNA activity across cell lines, robustly identifies inefficient gRNAs across different published datasets, and is easily accessible via online genome browser tracks. In addition, we develop a homology-directed repair efficiency prediction tool and show that unintended large-scale repair events are common for Cas9 but not for Cas12a, which may be relevant for safety in gene therapy applications.

Cell-tagging strategies with DNA barcodes have enabled the analysis of clone size dynamics and clone-restricted transcriptomic landscapes in heterogeneous populations. However, isolating a target clone that displays a specific phenotype from a complex population remains challenging. Here we present a multi-kingdom genetic barcoding system, CloneSelect, which enables a target cell clone to be triggered to express a reporter gene for isolation through barcode-specific CRISPR base editing. In CloneSelect, cells are first stably tagged with DNA barcodes and propagated so that their subpopulation can be subjected to a given experiment. A clone that shows a phenotype or genotype of interest at a given time can then be isolated from the initial or subsequent cell pools stored during the experiment using CRISPR base editing. CloneSelect is scalable and compatible with single-cell RNA sequencing. We demonstrate the versatility of CloneSelect in human embryonic kidney 293T cells, mouse embryonic stem cells, human pluripotent stem cells, yeast cells and bacterial cells.

Diffuse midline glioma (DMG) is a fatal childhood brain tumor characterized primarily by mutant histone H3 (H3K27M). H3K27M causes a global reduction in Polycomb repressive complex 2 (PRC2)-mediated H3K27 trimethylation (H3K27me3). Paradoxically, PRC2 is essential in DMG cells, although the downstream molecular mechanisms are poorly understood. Here, we have discovered a specific form of canonical PRC1 (cPRC1) containing CBX4 and PCGF4 that drives oncogenic gene repression downstream of H3K27me3 in DMG cells. Via a novel functional region, CBX4 preferentially associates with PCGF4-containing cPRC1. The characteristic H3K27me3 landscape in DMG rewires the distribution of cPRC1 complexes, with CBX4/PCGF4-cPRC1 accumulating at H3K27me3-enriched CpG islands. Despite comprising <5% of cPRC1 in DMG cells, the unique repressive functions of CBX4/PCGF4-cPRC1 are essential for DMG growth. Our findings link the altered distribution of H3K27me3 to imbalanced cPRC1 function, which drives oncogenic gene repression in DMG, highlighting potential therapeutic opportunities for this incurable childhood brain cancer.

Wilms tumor is the most common pediatric kidney cancer, and diffuse anaplastic Wilms tumor is the most chemoresistant subtype. Here, we explore how Wilms tumor cells evade the chemotherapy actinomycin D, which inhibits ribosomal RNA biogenesis. Using ribosome profiling, protein arrays, and a genome-wide knockout screen, we describe how actinomycin D disrupts protein homeostasis and blocks cell-cycle progression. When ribosomal capacity is limited by actinomycin D treatment, anaplastic Wilms tumor cells preferentially translate proteasome components. Next, we find that the proteasome inhibitor bortezomib sensitizes cells to actinomycin D treatment in vitro and prolongs survival in xenograft models. Lastly, increased levels of proteasome components are associated with anaplastic histology and worse prognosis in Wilms tumor patients. In sum, maintaining protein homeostasis is critical for Wilms tumor proliferation, and it can be therapeutically disrupted by blocking protein synthesis or turnover.

Gynecological cancers, including cervical, ovarian, and endometrial cancers, represent a significant global health challenge due to their high prevalence and profound impact on mortality and quality of life. This narrative review explores the transformative capability of genome editing, specifically clustered regularly interspaced short palindromic repeats (CRISPR/Cas9) technology, in advancing the management of these cancers. Genome editing offers great opportunities to develop targeted therapies by enabling precise modifications of genes involved in cancer initiation, progression, and chemoresistance.

A comprehensive literature search was conducted from October 2004 to October 2024. Only peer-reviewed relevant English articles with substantial insights into the impact of genome editing on cancer therapies were considered using keywords such as "CRISPR/Cas9," "genome editing," "gynecological cancers," and specific cancer types like "cervical cancer," "ovarian cancer," and "endometrial cancer."

Genome editing, particularly CRISPR/Cas9, holds substantial capacity for revolutionizing the treatment landscape of gynecological cancers by enabling highly specific, gene-targeted therapies that can overcome conventional treatment limitations such as chemoresistance and tumor recurrence. Emerging preclinical studies demonstrate the feasibility of correcting oncogenic mutations and enhancing the sensitivity of tumors to existing therapies. However, before clinical translation can be realized, critical challenges-including off-target effects, delivery system optimization, and immune responses-must be systematically addressed through rigorous research and clinical trials. Advancing these solutions will be essential for safely integrating CRISPR-based interventions into personalized medicine approaches for gynecological malignancies.

Respiratory viruses represent a major global health burden. Although these viruses have different life cycles, they may depend on common host genetic factors, which could be targeted by broad-spectrum host-directed therapies. We used genome-wide CRISPR screens and advanced data analytics to map a network of host genes that support infection by nine human respiratory viruses [influenza A virus, parainfluenza virus, human rhinovirus, respiratory syncytial virus, human coronavirus (HCoV)-229E, HCoV-NL63, HCoV-OC43, Middle East respiratory syndrome-related coronavirus, and severe acute respiratory syndrome-related coronavirus 2]. We explored shared pathways using knowledge graphs to inform on pharmacological targets. We selected and validated STT3A/B proteins of the N-oligosaccharyltransferase complex as host targets of broad-spectrum antiviral small molecules. Our work highlights the commonalities of viral host genetic dependencies and the feasibility of using this information to develop broad-spectrum antiviral agents.

Single-cell RNA-seq (scRNAseq) struggles to capture the cellular heterogeneity of transcripts within individual cells due to the prevalence of highly abundant and ubiquitous transcripts, which can obscure the detection of biologically distinct transcripts expressed up to several orders of magnitude lower levels. To address this challenge, here we introduce single-cell CRISPRclean (scCLEAN), a molecular method that globally recomposes scRNAseq libraries, providing a benefit that cannot be recapitulated with deeper sequencing. scCLEAN utilizes the programmability of CRISPR/Cas9 to target and remove less than 1% of the transcriptome while redistributing approximately half of reads, shifting the focus toward less abundant transcripts. We experimentally apply scCLEAN to both heterogeneous immune cells and homogenous vascular smooth muscle cells to demonstrate its ability to uncover biological signatures in different biological contexts. We further emphasize scCLEAN's versatility by applying it to a third-generation sequencing method, single-cell MAS-Seq, to increase transcript-level detection and discovery. Here we show the possible utility of scCLEAN across a wide array of human tissues and cell types, indicating which contexts this technology proves beneficial and those in which its application is not advisable.

Phytoplankton are responsible for half of the global photosynthesis and form vast blooms in aquatic ecosystems. Bloom demise fuels marine microbial life and is suggested to be mediated by programmed cell death (PCD) induced by diverse environmental stressors. Despite its importance, the molecular basis for algal PCD remains elusive. Here, we reveal novel PCD genes conserved across distant algal lineages using cell-to-cell heterogeneity in the response of the diatom Phaeodactylum tricornutum to oxidative stress. Comparative transcriptomics of sorted sensitive and resilient subpopulations following oxidative stress revealed genes directly linked to their contrasting fates of cell death and survival. Comparing these genes with those found in a large-scale mutant screen in the green alga Chlamydomonas reinhardtii identified functionally relevant conserved PCD gene candidates, including the cysteine protease cathepsin X/Z (CPX). CPX mutants in P. tricornutum CPX1 and C. reinhardtii CYSTEINE ENDOPEPTIDASE 12 (CEP12) exhibited resilience to oxidative stress and infochemicals that induce PCD, supporting a conserved function of these genes in algal PCD. Phylogenetic and predictive structural analyses show that CPX is highly conserved in eukaryotes, and algae exhibit strong structural similarity to human Cathepsin X/Z (CTSZ), a protein linked to various diseases. CPX is expressed by diverse algae across the oceans and correlates with upcoming demise events during toxic Pseudo-nitzschia blooms, providing support for its ecological significance. Elucidating PCD components in algae sheds light on the evolutionary origin of PCD in unicellular organisms and on the cellular strategies employed by the population to cope with stressful conditions.

Corylus species (hazelnuts) are a valuable source of nutrients and are widely consumed worldwide. Nevertheless, Corylus avellana (Cor a) contains 13 allergens (Cor a 1, Cor a 2, Cor a 6, Cor a 8, Cor a 9, Cor a 10, Cor a 11, Cor a 12, Cor a 13, Cor a 14, Cor a 15, Cor a 16, and Cor a TLP) that have been deposited into the official database (WHO/IUIS) for allergen nomenclature. The recent availability of several Corylus genomes provided opportunities to explore allergome variability, and thus to develop hypoallergenic varieties using modern biotech approaches. Certainly, the identification of CRISPR-Cas9 guide RNA (gRNA) is a pivotal step in achieving this goal. User-friendly web tools include limited reference genomes to design CRISPR-Cas9 gRNAs, while bioinformatic software for custom analysis require advanced command-line skills.

This work explored the evolutionary trajectories of allergenic Cor a homologs in C. avellana, C. americana, C. heterophylla, and C. mandshurica genome assemblies. 52 Cor a orthologs were found in the analyzed species, and a recent tandem duplication of Cor a 1 was found in C. americana. Three new gene models were predicted in C. avellana and C. mandshurica for Cor a 16 and Cor a 10. Additionally, we identified 56 Cor a isoallergens, of which ten Cor a isoforms. Furthermore, phylogenetic analysis sheds light on the evolutionary dynamics of three hazelnut allergens revealing the evolutionary complexity of Cor a 1, Cor a 2, and Cor a TLP within the Corylus genus. A list of multiple gRNAs designed for the CRISPR-Cas9 system was provided for the singular and multiple silencing of Cor a homologs in each Corylus genome.

This study enhances our knowledge on the evolutionary path of Cor a allergens among Corylus species and provides highly accurate on-target guides targeting hazelnut allergome.

Calcium signals regulate crucial cellular functions yet many genes coding for Ca2+handling proteins remain unknown as their identification relies on low-throughput single-cell approaches. Here we describe a method to measure Ca2+ activity using CaMPARI2, flow cytometry and pooled genome interrogation. CAMPARI2 screen (CaMP-Screen) identified enhancers and inhibitors of homeostatic Ca2+ activity, highlighting a predominant role for store-operated Ca2+ entry (SOCE) and lipid signalling pathways. Genes reducing basal Ca2+ activity were linked to Prader Willy syndrome, T cell dysfunction, and deafness. Silencing of HAVCR1 gene, coding for T cell transmembrane immunoglobulin and mucin (TIM1), enhanced Ca2+ signals in T cells and promoted signaling under resting but not after TCR engagement. Our findings establish CaMP-Screen as an efficient detector of low-amplitude Ca2+ signals and identify new genes associated to pathologies that regulate Ca2+ homeostasis, reporting TIM1 as a negative regulator of Ca2+ signals driving T cell function.

During cortical development, neural stem/precursor cells (NS/PCs) sequentially produce neurons, astrocytes, and oligodendrocytes. Before producing these cells, human (h) NS/PCs undergo prolonged self-renewal to form a larger cortex than other mammals, although the mechanisms are mostly unknown. Here, we performed a gene knockout screen using the CRISPR/Cas9 system to search for genes involved in hNS/PC self-renewal. We identified RMND5A, encoding an E3 ubiquitin ligase, among the candidate genes. We further demonstrated that knockdown of RMND5A decreased proliferation and promoted neuronal differentiation of hNS/PCs through the activation and suppression of the Wnt and mTOR signaling pathways, respectively. Taken together, our findings suggest that RMND5A participates in the maintenance of hNS/PC self-renewal by modulating the Wnt and mTOR signaling pathways. Impact statement During cortical development, human neural stem/precursor cells (hNS/PCs) undergo prolonged self-renewal to form a larger cortex than other mammals, although the mechanisms are mostly unknown. We identified RMND5A, an E3 ubiquitin ligase, as essential for maintaining self-renewal of hNS/PCs, providing valuable insights into the evolutionary expansion of the human brain.

CRISPR-dependent base editing (BE) enables the modeling and correction of genetic mutations at single-base resolution. Base editing screens, where point mutations are queried en masse, are powerful tools to systematically draw genotype-phenotype associations and characterise the function of genes and other genomic elements. However, the lack of user-friendly web-based tools for designing base editing screens can hinder broad technology adoption. Here, we introduce CRISPR-BEasy (https://crispr-beasy.cerc-genomic-medicine.ca), a free, automated web-based server that streamlines the creation of single guide (sg)RNA tiling libraries for base editing screens. Researchers can provide their genes or genomic features of interest, their base editors of choice, and target sequences to act as positive and negative controls. The server designs and annotates sgRNA libraries by integrating custom code with publicly available tools such as crisprVerse and Ensembl's Variant Effect Predictor. CRISPR-BEasy provides downloadable results, including sgRNA on/off-target scores, predicted mutational outcomes per base editor, and intuitive interactive visualizations for data quality assessment. CRISPR-BEasy also provides a separate tool that assembles sgRNA libraries into oligonucleotides for cloning following the detailed protocol documented in the searchable web server manual. Together, CRISPR-BEasy ensures the seamless design of cloning-ready sgRNA libraries, seeking to democratise access to base editing screening technologies.

Identifying beneficial or functional mutations for a specific species is a task always relies on the labor-intensive construction of a library containing thousands of single-gene knockout or interference strains. Here, we systematically demonstrated that the task could be done by constructing a genome-scale library, designed by AutoGSL, containing a limited number of large fragment deletion strains. The loss-of- and gain-of-function phenotypes could be efficiently screened out by our chromosome-segment scanning, and the specific gene corresponding to an observed phenotype could be quickly identified and visualized by our computer-aided gene-annotation web tool. Additionally, a Clusters of Orthologous Gene Transformer learned representations were transferred to predict growth phenotypes of the genome-scale library under varying conditions. We further utilized our chromosome segment scanning for gain- or loss-of-function screening (CHASING) strategy to obtain acetoin- and lycopene-overproducing hosts. Our work highlighted the significance of CHASING in functional genomics investigation, robust chassis engineering, and chemical overproduction.

Pancreatic cancer is characterized by late diagnosis, therapy resistance, and poor prognosis, necessitating the exploration of early carcinogenesis and prevention methods. Preclinical mouse models have evolved from cell line-based to human tumor tissue- or organoid-derived xenografts, now to humanized mouse models and genetically engineered mouse models (GEMMs). GEMMs, primarily driven by oncogenic Kras mutations and tumor suppressor gene alterations, offer a realistic platform for investigating pancreatic cancer initiation, progression, and metastasis. The incorporation of inducible somatic mutations and CRISPR-Cas9 screening methods has expanded their utility. To better recapitulate tumor initiation triggered by inflammatory cues, common pancreatic risk factors are being integrated into model designs. This approach aims to decipher the role of environmental factors as secondary or parallel triggers of tumor initiation alongside oncogenic burdens. Emerging models exploring pancreatitis, obesity, diabetes, and other risk factors offer significant translational potential. This review describes current mouse models for studying pancreatic carcinogenesis, their combination with inflammatory factors, and their utility in evaluating pathogenesis, providing guidance for selecting the most suitable models for pancreatic cancer research.

DNA methyltransferase 3A (DNMT3A) plays crucial roles in hematopoiesis and mammalian development. DNMT3A protein instability has been associated with several diseases such as MDS, AML and Tatton-Brown-Rahman syndrome. Here we report, DNMT3A stability is maintained by deubiquitinating enzyme USP11 countering degradation by CUL4-DCAF8 E3 ligase. DNMT3A localization changes caused by certain unstable DNMT3A mutations, which could be considered one of the losses of function of DNMT3A. The mislocalization is partially rescued by E1 enzyme inhibition or stable USP11 expression lines. Interestingly, we show that USP11 enhances DNMT3A SUMOylation by promoting the interaction between DNMT3A and SUMO E3 Ligases, and DNMT3A SUMOylation also essential for maintaining DNMT3A protein stability and DNMT3A DNA Mtase activity. Our results reveal the mechanism for DNMT3A protein turnover through USP11, and the mechanism essential for DNMT3A function, as well as a therapeutic approach for several diseases causing DNMT3A protein instability.

Registered systemic treatment options for glioblastoma patients are limited. The phase II REGOMA trial suggested an improvement of median overall survival in progressive glioblastoma by the multi-tyrosine kinase inhibitor regorafenib. This has not been confirmed by GBM AGILE. So far, regorafenib has been administered as monotherapy or as an addition to standard of care in newly diagnosed glioblastoma. Rational combination therapies involving regorafenib might be a reasonable strategy. Here, we aimed at identifying functionally instructed combination therapies involving regorafenib.

We applied a genome-wide CRISPR-Cas9-based functional genomics target discovery approach using activation and knockout screens followed by genetic, pharmacological, functional validations. Regorafenib-induced molecular alterations were assessed by RNA sequencing and DigiWest. We investigated selected functionally instructed combination therapies in three orthotopic glioma mouse models in vivo (syngeneic SMA560/VM/Dk model and two xenograft models) and performed immunohistochemistry of post-treatment brains.

We identified potential modifiers of regorafenib response, including BCL2, BCL2L1, ITGB3, FOXC1, SERAC1, ARAF, and PLCE1. The combination of regorafenib with Bcl-2/Bcl-xL inhibition was superior to both monotherapies alone in vitro, ex vivo, and in vivo. We identified regorafenib-induced regulations of the Bcl-2 downstream target chemokine receptor 1 (CCR1) as one potential underlying molecular mediator. Furthermore, regorafenib led to changes in the myeloid compartment of the glioma-associated microenvironment.

This preclinical study uses a functional genomics-based target discovery approach with subsequent validations involving regorafenib. It serves as a biological rationale for clinical translation. Particularly, an investigation of the combination of regorafenib plus navitoclax within a clinical trial is warranted.

DNA methyltransferase 1 (DNMT1) is an enzyme known for DNA methylation maintenance. Point mutations in its replication focus targeting sequence (RFTS) domain lead to late-onset neurodegeneration, such as autosomal dominant cerebellar ataxia-deafness and narcolepsy (ADCA-DN) disorder. Here, we demonstrated that DNMT1 has the capability to bind to mRNA transcripts and facilitate 5-methylcytosine (m5C) RNA methylation by recruiting NOP2/Sun RNA methyltransferase 2 (NSUN2). RNA m5C methylation, in turn, promotes RNA stability for those genes modulating mitochondrial function. When the DNMT1 RFTS domain is mutated in mice, it triggers aberrant DNMT1-RNA interaction and significantly elevated m5C RNA methylation and RNA stability for a portion of metabolic genes. Consequently, increased levels of metabolic RNA transcripts contribute to cumulative oxidative stress, mitochondrial dysfunction, and neurological symptoms. Collectively, our results reveal a dual role of DNMT1 in regulating both DNA and RNA methylation, which further modulates mitochondrial function, shedding light on the pathogenic mechanism of DNMT1 mutation-induced neurodegeneration.

The TAL1 oncogene driving T cell lymphoblastic leukemia is frequently activated through mutated cis-regulatory elements, whereby small insertions or deletions (indels) create a binding site for the transcription factor MYB. Unraveling how non-coding mutations create oncogenic enhancers is key to understanding cancer biology and can provide important insights into fundamental mechanisms of gene regulation. Utilizing a CRISPR-Cas9 screening approach, we identify GATA3 as the key transcriptional regulator of enhancer-mediated TAL1 overexpression. CRISPR-Cas9 engineering of the mutant enhancer reveals a tandem GATA3 site that is required for binding of GATA3, chromatin accessibility, and MYB recruitment. Reciprocally, MYB binding to its motif is required for GATA3 recruitment, consistent with a transcription factor cooperativity model. Importantly, we show that GATA3 stabilizes a TAL1-MYB interaction and that complex formation requires GATA3 binding to DNA. Our work sheds light on the mechanisms of enhancer-mediated oncogene activation, where key transcription factors cooperate to achieve maximal transcriptional output, thereby supporting leukemogenesis.

Esophageal cancer (EC) is the eighth most common cancer and the sixth most common cause of death worldwide. Esophageal squamous cell carcinoma (ESCC) comprises the majority of esophageal cancers globally, and 5-Fluorouraci (5-FU) is one of the commonly used chemotherapeutics for this type of cancer. Chemoresistance to drugs is a main obstacle in the successful treatment of this malignancy.

In this study, we used the CRISPR/Cas9 screening method to determine the target gene related to 5-FU drug resistance in esophageal cancer.

Our research findings indicate that the loss of PFKFB3 can increase the resistance of different human esophageal squamous cell carcinoma cell lines to 5-FU through various pathways. Specifically, in KYSE-70 cells, loss of PFKFB3 can induce epithelial-mesenchymal transition (EMT) and prolong the S phase of the cell cycle, allowing cancer cells to evade the effects of 5-FU and develop resistance. In the KYSE-270 and KYSE-150 cell lines, loss of PFKFB3 can upregulate the expression of Slug and Mcl-1, indirectly regulate Chk1 and promote its autophosphorylation, which in turn inhibits apoptosis, thus counteracting the effects of 5-FU.

Our research not only enriches our understanding of the biological characteristics of different ESCC cell lines but also provides new clinical insights for future personalized treatments. Assessing the status of PFKFB3 can help predict resistance to 5-FU in ESCC patients with different genetic backgrounds, allowing for more precise treatment planning. This personalized approach has the potential to improve treatment efficacy, reduce unnecessary drug use and side effects, and ultimately improve patient survival rates and quality of life.

Quiescence cancer stem-like cells may play key roles in promoting tumor cell heterogeneity and recurrence for many tumors, including glioblastoma (GBM). Here we show that the protein acetyltransferase KAT5 is a key regulator of transcriptional, epigenetic, and proliferative heterogeneity impacting transitions into G0-like states in GBM. KAT5 activity suppresses the emergence of quiescent subpopulations with neurodevelopmental progenitor characteristics, while promoting GBM stem-like cell (GSC) self-renewal through coordinately regulating E2F- and MYC- transcriptional networks with protein translation. KAT5 inactivation significantly decreases tumor progression and invasive behavior while increasing survival after standard of care. Further, increasing MYC expression in human neural stem cells stimulates KAT5 activity and protein translation, as well as confers sensitivity to homoharringtonine, to similar levels to those found in GSCs and high-grade gliomas. These results suggest that the dynamic behavior of KAT5 plays key roles in G0 ingress/egress, adoption of quasi-neurodevelopmental states, and aggressive tumor growth in gliomas.