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Results for “CRISPR knockout”

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At least 19 recordsLinked to original sources

PubMed2026

Aneuploidy selects for the acquisition of driver genes in breast cancer.

Chromosome instability is highly prevalent in cancer and drives large-scale chromosomal imbalances, known as aneuploidies1-4. How aneuploidy contributes to tumorigenesis remains difficult to study due to the vast numbers of genes affected. Here we established a CRISPR knockout- and activation-linked assay (CRISPR-KOALA), enabling high-throughput bidirectional genetic screens in immunocompetent mouse models of cancer. We developed a compendium of the ten most frequent human chromosome-arm-level alterations in basal-like breast cancer (BLBC), a disease type that is driven by large copy-number alterations (CNAs)5-8. Using CRISPR-KOALA, we screened the mouse orthologues of 3,752 genes on these arms and identified 90 cancer driver genes, the function of the vast majority of which is unknown. These genes drive distinct signalling pathways including MAPK, HIPPO and WNT, reflecting the high degree of BLBC heterogeneity. Manipulating the identified cancer driver genes overcomes the need for CNAs in Trp53-mutant BLBC mouse models. Mechanistically, we identify that PLGRKT is a potent oncogene that lies on chromosome 9p and show that its tumour-promoting activity is associated with highly stress-resistant mitochondria and an increased ability to detoxify reactive oxygen species. Together, our findings reveal that arm-level CNAs can function to select specific driver genes to promote heterogeneous biological processes.

Animals↗

PubMed2025

CRISPR-Cas9-mediated knockout of OsKCS11 in rice reveals potential crosstalk between very-long-chain fatty acids and cytokinin.

Very-long-chain fatty acids (VLCFAs) play crucial roles in various physiological processes in plants. Through our investigation using a CRISPR-Cas9 knockout mutant library in rice, we identified a semi-dwarf rice mutant named CRISPR-Cas-based dwarf-1 (csd-1). This mutant displayed multiple developmental defects, such as decreased plant height, panicle length, seed size, and seed-setting rate. Whole-genome resequencing analysis revealed that a T-nucleotide insertion in β-ketoacyl-CoA synthase 11 (KCS11), responsible for the initial step in fatty acid elongation, was responsible for the observed defects in csd-1. The identity of csd-1 was confirmed through genetic complementation and CRISPR-Cas9-mediated knockout. Expression analysis indicated that OsKCS11 was present in various tissues, with differential abundance observed through RT-qPCR and promoter GUS staining, and strong localization at the node position by RNA in situ hybridization; furthermore, OsKCS11 protein was confirmed to be in the endoplasmic reticulum. Furthermore, csd-1 exhibited significantly reduced levels of linolenic acid (18:3), C24:0-OH, C28:0-alkanes, C29:0-alkanes, alpha-tocopherol, and C33:0-alkanes, while trans-nonadecenoic acid and behenic acid levels were increased. Cytokinin analysis revealed significant increases in isopentenyladenine (IPA) and cis-zeatin (cZ) levels in csd-1. Molecular investigations indicated upregulation of genes involved in cytokinin biosynthesis or signaling, suggesting a potential link between VLCFAs and cytokinin synthesis through acetyl-CoA. This study not only proposed an alternative gene mapping method based on whole-genome resequencing but also elucidated the mechanism by which VLCFAs influence cytokinin synthesis and signaling.

Oryza↗

PubMed2025

Tonoplast sucrose transporter SUT4-dependent sugar partitioning modulates phenological transitions and reproductive success in poplar.

Climate uncertainty is intensifying the need for greater plasticity in carbohydrate reserve utilization to support winter survival and spring growth in woody perennials. In poplar, the single-copy SUT4, which encodes a tonoplast-localized sucrose transporter, and the SUT5/SUT6 genome duplicates, which encode plasma membrane-localized transporters, are expressed year-round, with SUT4 showing the highest expression during cool seasons. Given its role in vacuolar sucrose efflux and winter-predominant expression, SUT4 may play a key role in modulating seasonal carbohydrate dynamics. While SUT4-knockdown and knockout effects have been studied under greenhouse conditions, their impact under field conditions remains unexplored. Here, we report a field-based study comparing CRISPR knockout mutants of winter-expressed SUT4 and SUT5/SUT6 in Populus tremula × alba. We show that sut4, but not sut5/6, mutants exhibited earlier autumn leaf senescence, delayed spring bud flush, reduced stem growth, and altered sugar partitioning in winter xylem and bark relative to controls. After 2 years in the field, all genotypes flowered before leaf flush in early spring; however, sut4 mutants produced sterile ovules despite developing normal-looking catkins. Metabolic profiling revealed disrupted sucrose and raffinose dynamics in elongating sut4 catkins. This was accompanied by transcriptomic signatures of elevated stress and downregulation of proanthocyanidin biosynthesis and circadian clock genes. These findings highlight the critical role of SUT4 in coordinating sugar allocation, stress responses, and seasonal development in poplar.

Populus↗

PubMed2025

UHRF1 restricts HCoV-229E infection through epigenetic silencing of the viral receptor APN.

The emergence of SARS-CoV-2 has posed significant threats to global health, particularly for the older population. Similarly, common human coronaviruses, such as HCoV-229E, which typically cause mild cold-like symptoms, can lead to severe diseases, underscoring the need to understand virus-host interactions and identify host factors contributing to viral pathogenesis and disease progression. In this study, we perform a genome-wide CRISPR knockout screen using HCoV-229E and identify UHRF1 as a potent restriction factor. Mechanistically, UHRF1 suppresses HCoV-229E infection by downregulating the expression of its cell entry receptor, APN, through promoter hypermethylation. Focused CRISPR activation screens of UHRF1-downregulated genes confirm the critical role of APN in HCoV-229E infection and identify additional genes (e.g., SIGLEC1, PLAC8, and heparan sulfate biosynthesis genes) contributing to the restrictive functions of UHRF1. Transcriptomic and single-cell RNA sequencing analysis reveal that UHRF1 expression decreases with age, negatively correlating with increased APN expression. This age-related decline in UHRF1 is validated in primary alveolar macrophages from elderly individuals, which exhibit heightened susceptibility to HCoV-229E compared to those from younger individuals. Our findings highlight UHRF1 as a key age-related host defense factor against coronavirus and provide insights into the epigenetic regulation of viral entry receptors.

Animals↗

PubMed2025

In vivo genome-wide CRISPR screens in human T cells to enhance T cell therapy for solid tumors.

Large-scale CRISPR screening in human T cells holds significant promise for identifying genetic modifications that can enhance cellular immunotherapy. However, many genetic regulators of T cell performance in solid tumors may not be readily revealed in vitro. In vivo screening in tumor-bearing mice offers greater physiological relevance, but has historically been limited by low intratumoral T cell recovery. Here, we developed a new model system that achieves significantly higher human T cell recovery from tumors, enabling genome-wide in vivo screens with small numbers of mice. Tumor-infiltrating T cells in this model exhibit hallmarks of dysfunction compared to matched splenic T cells, creating an ideal context for screening for genetic modifiers of T cell activity in the tumor microenvironment. Using this platform, we performed two genome-wide CRISPR knockout screens to identify genes regulating T cell intratumoral abundance and effector function (e.g., IFN-γ production). The intratumoral abundance screen uncovered the P2RY8-Gα13 GPCR signaling pathway as a negative regulator of human T cell infiltration into tumors. The effector function screen identified GNAS (Gαs), a central signaling mediator downstream of multiple GPCRs that sense different suppressive ligands, as a key regulator of T cell dysfunction in tumors. Targeted GNAS knockout rendered T cells resistant to multiple suppressive cues and significantly improved therapeutic performance across diverse solid tumor models. Moreover, combinatorial knockout of P2RY8 (trafficking) and GNAS (effector function) further enhanced overall tumor control, demonstrating that genetic modifications targeting distinct T cell phenotypes can be combined to improve therapeutic potency. This flexible and scalable in vivo screening platform can be adapted to diverse tumor models and pooled CRISPR libraries, enabling future discovery of genetic strategies that equip T cell therapies to overcome barriers imposed by solid tumors.

Journal Article↗

PubMed2025

Defining the host dependencies and the transcriptional landscape of RSV infection and bystander activation.

Respiratory syncytial virus (RSV) is a globally prevalent pathogen, causes severe disease in older adults, and is the leading cause of bronchiolitis and pneumonia in the United States for children during their first year of life [1]. Despite its prevalence worldwide, RSV-specific treatments remain unavailable for most infected patients. Here, we leveraged a combination of genome-wide CRISPR knockout screening and single-cell RNA sequencing to improve our understanding of the host determinants of RSV infection and the host response in both infected cells, and uninfected bystanders. These data reveal temporal transcriptional patterns that are markedly different between RSV infected and bystander activated cells. Our data show that expression of interferon-stimulated genes is primarily observed in bystander activated cells, while genes implicated in the unfolded protein response and cellular stress are upregulated specifically in RSV infected cells. Furthermore, genome-wide CRISPR screens identified multiple host factors important for viral infection, findings which we contextualize relative to 29 previously published screens across 17 additional viruses. These unique data complement and extend prior studies that investigate the proinflammatory response to RSV infection, and juxtaposed to other viral infections, provide a rich resource for further hypothesis testing.

CRISPR screen↗

PubMed2026

Genome-wide CRISPR screen reveals PEX11B as a host restriction factor against ORFV through membrane fluidity regulation.

Host-pathogen interactions are shaped by cellular restriction factors that direct antiviral defenses. We built the first ovine genome-wide CRISPR knockout library in sheep testis (OA3.Ts) cells, targeting all protein-coding genes. Using this platform, we identified PEX11B, a peroxisomal membrane regulatory protein, as a strong restriction factor against orf virus (ORFV) infection. Removing PEX11B increased viral susceptibility and triggered severe cytopathic effects with membrane fusion and syncytia formation. Mechanistic studies showed that PEX11B knockout harmed peroxisomal integrity and disrupted lipid metabolism. This led to greater plasma membrane fluidity, creating a proviral environment that allowed more viral entry and replication. These results reveal a new antiviral function for PEX11B in blocking viral infection and underscore the importance of peroxisomal regulation in host-virus interactions.

Animals↗

PubMed2025

RESTRICT-seq enables time-gated CRISPR screens and uncovers novel epigenetic dependencies of SCC resistance.

Cancer cell evasion of therapy is a highly adaptive process that undermines the efficacy of many treatment strategies. A significant milestone in the study of these mechanisms has been the advent of pooled CRISPR knockout screens, which enable high-throughput, genome-wide interrogations of tumor dependencies and synthetic lethal interactions, advancing our understanding of how cancer cells adapt to and evade therapies. However, the utility of this approach diminishes when applied to dynamic biological contexts, where processes are transient and sensitivity to routine cell culture manipulations that introduce noise and limit meaningful discoveries. To overcome these limitations, we present RESTRICT-seq, a next-generation pooled screening methodology that restricts Cas9 nuclear activation in controlled, repeated cycles. By confining Cas9 catalytic activity to strict temporal windows, RESTRICT-seq mitigates undesired fitness penalties that routinely accumulate throughout pooled screens. When benchmarked against conventional pooled screens and standard inducible protocols, RESTRICT-seq revealed significantly fewer divergent cell clones and increased signal-to-noise ratio, overcoming a key limitation of traditional methods. Leveraging RESTRICT-seq, we conducted a comprehensive functional survey of the druggable mammalian epigenome, uncovering several elusive epigenetic drivers of treatment resistance in cutaneous squamous cell carcinoma (cSCC). This revealed PAK1 as a previously unrecognized mediator of cSCC resistance in human and mouse SCC, offering new insights into a prognostic marker and therapeutic target of high clinical significance. Our findings establish RESTRICT-seq as a powerful tool for extending the applicability of pooled CRISPR screens to dynamic and previously intractable biological contexts.

Allosterically-regulated Cas9 (arCas9)↗

PubMed2026

CRISPR Screen Identifies HDAC3 as a Novel Radiosensitizing Target in Small Cell Lung Cancer.

Small cell lung cancer (SCLC) is an aggressive malignancy, with most patients presenting with prognostically poor extensive-stage disease. Limited progress in standard care stresses the urgent need for novel therapies. Radiotherapy offers some survival benefit for selected patients with SCLC but could be enhanced with radiosensitizers. In this study, we identify HDAC3 as a novel radiosensitizing target in SCLC using a CRISPR knockout screen and demonstrate its efficacy and mechanism. SBC5 cells were transduced with a custom EpiDrug single-guide RNA library and treated with ionizing radiation (IR) to identify radiosensitizing genes. HDAC3 emerged as a candidate and was validated through genetic knockdown and pharmacologic inhibition (RGFP966) in multiple SCLC cell lines. Both approaches enhanced radiosensitivity, as shown by cell viability (dose modification factor10 = 1.14-1.69) and clonogenic assays (dose modification factor10 = 1.16-1.41). We assessed changes in chromatin accessibility by assay for transposase-accessible chromatin using sequencing and IR-induced DNA damage and repair using γH2AX foci detection, double-strand break (DSB) repair assays, and immunoblotting of repair proteins. HDAC3-deficient cells exhibited increased chromatin accessibility, greater IR-induced DSBs, and impaired repair capacity, resulting in persistent DNA damage. This repair defect sensitized cells to PARP inhibitors, for which combining RGFP966 with olaparib or talazoparib produced additive to synergistic effects. In SCLC xenograft models, HDAC3 knockdown or RGFP966, combined with IR, achieved significant tumor growth inhibition. Collectively, we identified HDAC3 as a novel radiosensitizing target in SCLC. Its functional loss increased the generation and persistence of IR-induced DNA DSBs, effectively sensitizing SCLC cell lines and xenografts to IR, providing a potential radiosensitization strategy to treat SCLC.

Humans↗

PubMed2026

Comprehensive In Silico Analysis Identifies MSTO1 and LIG1 as Candidate Biomarkers With Diagnostic and Prognostic Relevance in Hepatocellular Carcinoma.

BACKGROUND: Hepatocellular carcinoma (HCC) is the most common primary liver malignancy and remains a major cause of cancer-related mortality worldwide. Its poor clinical outcomes are largely attributed to late-stage diagnosis and the limited accuracy of currently available diagnostic and prognostic biomarkers. Therefore, identifying novel molecular markers with improved sensitivity, specificity, and therapeutic relevance is essential for enhancing early detection and guiding personalized treatment strategies. AIMS: To identify and prioritize novel candidate HCC biomarkers with diagnostic and prognostic value and potential therapeutic vulnerability using integrated multi-omics, survival, functional dependency, and tumor microenvironment analyses. METHODS AND RESULTS: We examined the mRNA and protein expression levels of 8 DEGs in HCC tissues in the TCGA and CPTAC datasets using UALCAN, which showed that MSTO1 and LIG1 were overexpressed consistently in HCC relative to normal liver tissues. Moreover, elevated expression levels of these genes were significantly associated with higher tumor grade and advanced stage. Kaplan-Meier plotter survival data confirmed that increased expression of MSTO1 and LIG1 was associated with poorer overall survival. The DepMap CRISPR knockout data confirmed a functional dependency of both genes in HCC cell lines. CBioPortal analyses provided characterization of genomic alterations and enabled enrichment analysis of co-expressed genes, and the TCGA-UALCAN pan-cancer analyses supported the assessment of tissue specificity across tumor types. TIMER3 analyses linked candidate gene expression with immune cell infiltration patterns. Diagnostic performance by ROC analysis showed excellent discrimination for MSTO1 (AUC = 0.987) and good discrimination for LIG1 (AUC = 0.897). Multivariate Cox regression with Benjamini-Hochberg FDR correction across the eight genes supported MSTO1 as a candidate independent prognostic factor after adjustment for tumor stage, grade, etiology, age, and sex (HR = 1.29, p = 0.035), whilst LIG1 showed no independent prognostic value. Promoter methylation of MSTO1 and ADH4, assessed via UALCAN, showed that both genes were significantly differentially methylated in the promoter region of primary HCC tissues compared with normal liver tissues. Our study also confirmed the biological and clinical relevance of established HCC biomarkers: TERT, IRAK1, and ADH4. CONCLUSION: MSTO1 and LIG1 emerged as candidate diagnostic biomarkers in HCC. Additionally, MSTO1 showed a candidate prognostic association with overall survival that remained significant after adjusting for tumor stage, grade, and etiology, as well as patients' age, but not after further adjustment for AFP status. Functional data also highlighted MSTO1 as a candidate therapeutic dependency. On the other hand, LIG1 showed no independent prognostic association in either multivariate model. Their differential expression and functional essentiality in HCC cell lines highlighted their value for further experimental and independent-cohort validation before potential integration into biomarker development pipelines aimed at improving early detection and targeted therapy in HCC.

Humans↗

PubMed2022

Genome-wide bidirectional CRISPR screens identify mucins as host factors modulating SARS-CoV-2 infection.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes a range of symptoms in infected individuals, from mild respiratory illness to acute respiratory distress syndrome. A systematic understanding of host factors influencing viral infection is critical to elucidate SARS-CoV-2-host interactions and the progression of Coronavirus disease 2019 (COVID-19). Here, we conducted genome-wide CRISPR knockout and activation screens in human lung epithelial cells with endogenous expression of the SARS-CoV-2 entry factors ACE2 and TMPRSS2. We uncovered proviral and antiviral factors across highly interconnected host pathways, including clathrin transport, inflammatory signaling, cell-cycle regulation, and transcriptional and epigenetic regulation. We further identified mucins, a family of high molecular weight glycoproteins, as a prominent viral restriction network that inhibits SARS-CoV-2 infection in vitro and in murine models. These mucins also inhibit infection of diverse respiratory viruses. This functional landscape of SARS-CoV-2 host factors provides a physiologically relevant starting point for new host-directed therapeutics and highlights airway mucins as a host defense mechanism.

Animals↗

PubMed2026

A HUWE1 regulatory helix gates ASCL1 degradation through its C-terminal phospho-degron in small cell lung cancer.

Lineage-defining transcription factors are key oncogenic drivers but remain difficult to target pharmacologically due to the absence of ligandable pockets. The molecular rules governing substrate recognition by large HECT ubiquitin ligases also remain incompletely understood, limiting efforts to exploit these enzymes for targeted protein degradation. Here we combine genome-wide CRISPR knockout screening with base editor tiling screens at amino acid resolution, both coupled to an endogenous knock-in reporter of the SCLC lineage oncogenic transcription factor ASCL1, to systematically interrogate the mechanisms governing its degradation. These complementary screens unbiasedly identify the HECT ubiquitin ligase HUWE1 as the dominant regulator of ASCL1 stability in small cell lung cancer (SCLC) and resolve a conserved C-terminal phospho-degron centered on Ser207 and terminal Trp/Phe residues that are required for HUWE1 docking and ubiquitin-mediated degradation. Unexpectedly, base editor screening further uncovers a previously unrecognized regulatory module within HUWE1: a short negatively charged helix that functions as an autoinhibitory gate controlling access of phospho-degron substrates to HUWE1. Charge-flipping mutations within this regulatory helix relieve autoinhibition and accelerate degradation of multiple HUWE1 phospho-degron substrates, including ASCL1 and the canonical HUWE1 substrate DDIT4. Stabilization of ASCL1 through degron disruption paradoxically impairs SCLC proliferation, revealing that dynamic proteasome-coupled turnover is required for transcription factor function. Together, these findings reveal molecular rules governing HUWE1 phospho-degron recognition and identify a regulatory gate controlling substrate engagement. They also illustrate a generalizable strategy for resolving degradation mechanisms of undruggable transcription factors in their endogenous cellular context.

ASCL1↗

PubMed2026

Identification of genomic features that uniquely impact estrogen receptor alpha binding and its effects on gene expression in endometrial cancer.

Estrogen receptor 1 (ESR1, also known as estrogen receptor alpha or ER) is an established oncogenic transcription factor in breast and endometrial cancer; however, more is known about the mechanisms controlling ER behavior in breast cancer, and therapies targeting ER have been much more successful in breast cancer. To address this disparity, we characterize the genomic features that control ER in endometrial cancer and determine to what extent these factors differ from those in breast cancer. We focus on the locations of estrogen response elements (EREs), ER's preferred DNA-binding motif, throughout the human genome. To identify factors that predict ER genomic binding and effects on target gene expression, we apply machine learning to genomic data for each ERE in Ishikawa cells (ER-positive endometrial cancer) and T-47D cells (ER-positive breast cancer). Many of these factors, such as chromatin accessibility and histone modifications, are predictive of ER activity in both cell lines. However, the transcription factors that predict ER activity are cell type specific, including FOXA1 and GATA3 in T-47D cells and ETV4 and SOX17 in Ishikawa cells. In addition, the features that predict ER binding and effects on gene expression differ, with transcription at EREs in the absence of estrogen being predictive of ER regulatory activity. A CRISPR knockout screen in Ishikawa cells, as well as follow-up experiments, confirms the discovery that SOX17 controls ER activity in endometrial cancer cells. These results identify important genomic features of ER binding and regulatory activity and how these features differ between endometrial cancer and breast cancer cells.

Humans↗

PubMed2026

Glutathione reductase deficiency potentiates the immunogenicity of ferroptosis and cuproptosis via amplified reactive oxygen species accumulation and cGAS-STING pathway activation.

BACKGROUND: Cancer remains a major therapeutic challenge due to drug resistance and metastasis, processes driven by oxidative stress and redox imbalance. Targeting this vulnerability through ferroptosis (iron-dependent lipid peroxidation) and cuproptosis (copper-driven mitochondrial dysfunction), two ROS-mediated cell death pathways, offers a promising therapeutic strategy. However, clinical translation is hindered by incomplete understanding of their redox regulation and limited immunogenicity. METHODS: A genome-wide CRISPR knockout screen was performed to identify key regulators of ferroptosis. Genetic depletion or pharmacological inhibition of candidate genes was evaluated across multiple cancer cell lines for sensitivity to ferroptosis inducer RSL3 and the cuproptosis inducer elesclomol (Es). Antitumor efficacy was assessed in xenograft, orthotopic, metastatic, and syngeneic mouse models, alone or combined with immune checkpoint inhibitors. Mechanistic studies also examined ROS production, mitochondrial stress, mitochondrial DNA release, cGAS-STING activation, and immune responses within the tumor microenvironment. RESULTS: Glutathione reductase (GSR), a central enzyme maintaining reduced glutathione (GSH) homeostasis, was identified as the top suppressor of ferroptosis. GSR knockout or pharmacological inhibition markedly sensitized diverse cancer cell lines to RSL3-induced ferroptosis, while GSR overexpression conferred resistance. Strikingly, GSR depletion also enhanced sensitivity to cuproptosis triggered by the copper ionophore Es. In multiple in vivo tumor models, GSR inhibition synergizes with RSL3 or Es to suppress tumor growth, inhibit lung metastasis, and prolong survival. Mechanistically, GSR deficiency amplified ROS production, induced mitochondrial stress, and triggered the cytosolic mitochondrial DNA release under ferroptotic or cuproptotic stress, activating the cGAS-STING pathway in vitro and in vivo. This increased inflammatory cytokine production, promoted immunogenic cell death, and enhanced the release of damage-associated molecular patterns (DAMPs), including HMGB1. Together, GSR inhibition combined with a ferroptosis or cuproptosis inducer transformed the tumor microenvironment into a highly immune stimulatory state, thereby enhancing the efficacy of immune checkpoint blockade through increased dendritic cell activation and T-cell infiltration and activation. CONCLUSIONS: GSR represents a key molecular node connecting and modulating ferroptosis and cuproptosis through redox regulation. Targeting GSR amplifies ROS-mediated immunogenic cell death, triggers cGAS-STING activation in cancer cells, and enhances the efficacy of cancer immunotherapy, providing a promising redox-based therapeutic strategy.

Ferroptosis↗

PubMed2026

Genome-wide association and multi-omics functional screens reveal the genetic architecture of foveal development.

Foveal hypoplasia causes visual impairment across congenital eye disorders, yet the genetic programmes governing foveal development remain poorly characterised and no tractable model exists for foveal disease. In the first genome-wide association study of foveal hypoplasia, we identified 42 sentinel variants mapping to 54 effector genes supported by ≥ 2 criteria from a variant-to-gene framework incorporating developmental multi-omics. Disruption of six effector genes using mutant lines and CRISPR knockouts in the zebrafish high acuity zone recapitulates structural, functional, and ultrastructural hallmarks of foveal hypoplasia, establishing the first vertebrate disease model. Integration with human foetal single-cell and spatial transcriptomics reveals two temporal waves of effector gene expression and identifies Müller glia as critical mediators of foveal patterning. Phenome-wide analyses reveal foveal variants are pleiotropic with refractive, lenticular, and metabolic traits, connecting foveal development to anterior segment and systemic disease biology. These findings should inform mechanistic studies of macular disease.

Journal Article↗

PubMed2025

CRISPR-based gene knockout in the model haloarchaeon Haloferax mediterranei.

Halophilic archaea, a specialized group of extremophiles that inhabit hypersaline environments, exhibit distinctive physiological and metabolic features. Traditional genetic manipulation of these organisms, predominantly reliant on homologous recombination techniques, suffers from limitations such as complex procedures and extended timelines, which hinder functional genomics research and the development of practical applications. This study established a CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)-mediated gene knockout system in the model halophilic archaeon Haloferax mediterranei. A polyethylene glycol (PEG)-mediated transformation method was used to deliver a plasmid carrying a mini-CRISPR array into H. mediterranei. The crtB gene, involved in pigment synthesis, was successfully knocked out, demonstrating the feasibility of CRISPR-based editing in H. mediterranei. To further validate the reliability and targeting accuracy of the system, the hlyR4 gene, encoding an extracellular serine protease, was also disrupted. The CRISPR-mediated gene knockout efficiency for hlyR4 reached 27%, significantly higher than the approximately 3% efficiency achieved with conventional homologous recombination. The establishment of this CRISPR-based gene knockout system provides a more efficient genetic tool for H. mediterranei and lays a new experimental foundation for exploiting microbial resources from extreme environments. In this study, H. mediterranei was selected as the model organism for haloarchaea. For the first time, we successfully constructed a CRISPR-based gene knockout system in a model halophilic archaeon. This system provides a solution for CRISPR-based gene knockout tools, which are currently unavailable in model halophilic archaea, and offers an effective tool for functional genomics studies in extremophiles.

Haloferax mediterranei↗

PubMed2026

A User-Friendly Protocol for Microinjection into Teleost Embryos to Study Gene Function.

Zebrafish (Danio rerio) and medaka (Oryzias latipes) are popular teleost models used in developmental biology and functional genomics. To achieve high-quality and reproducible microinjections, it is essential to have robust protocols for breeding, egg collection, and the precise delivery of genetic material. In this protocol, we present a comprehensive and optimized methodology for setting up breeding tanks under controlled photoperiod conditions to maximize egg yield while minimizing contamination. We provide detailed procedures for sex identification, pair selection, the use of grated breeding inserts, and methods to increase egg collection efficiency. We outline procedures for making injection gel beds, pulling needles, and calibration using one-microliter microcapillaries to achieve consistent nanoliter-scale injections. Our protocol outlines settings for the pico-liter injector that are optimized to deliver a precise amount per pulse with minimal variability. Finally, we demonstrate the application of these methods for gene knockdown using morpholino antisense oligonucleotides, gene knockout using CRISPR-Cas9, and gain-of-function mRNA overexpression experiments. Phenotypic assessments conducted at various developmental stages to evaluate gene-specific effects reveal consistent phenotypic outcomes between the morpholino and CRISPR-Cas9 approaches. This easy and comprehensive protocol enables efficient, precise, and scalable genetic manipulation of zebrafish and medaka embryos, thereby supporting advanced functional studies in developmental biology and disease modeling. To our knowledge, this is the first unified protocol for both zebrafish and medaka microinjection systems achieving 97.7% phenotype penetrance in CRISPR-Cas9 knockouts with precision together with a triple validation approach that confirms gene function across multiple techniques.

Animals↗

PubMed2025

Genome-wide CRISPR Screening Identifies NFκB and c-MET as Druggable Targets to Sensitize Lenvatinib Treatment in Hepatocellular Carcinoma.

BACKGROUND & AIMS: Hepatocellular carcinoma (HCC), the dominant form of liver cancer, is a leading cause of cancer death worldwide. Sorafenib and lenvatinib have long been the 2 limited options of first-line treatments for patients with unresectable advanced HCC. However, the single-drug treatment strategy only shows modest survival benefit, mostly because of the survival ability of cancer cells to activate alternative pathways for compensation. In this study, we aim to identify druggable targets contributing to lenvatinib resistance and evaluate the efficacy of combining respective inhibitors and lenvatinib on HCC. METHODS: Genome-scale clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 knockout library screening was applied on the vehicle group and lenvatinib treatment group. Identified druggable candidates were validated individually on HCC cell models. Therapeutic effects of the combined treatment of inhibitors of candidate genes and lenvatinib were evaluated in vitro and in vivo. RESULTS: We successfully identified NFKB1 and MET as critical drivers for the development of lenvatinib resistance in HCC cells. By perturbing the 2 genes with either CRISPR knockout or RNA interference approaches, lenvatinib treatments were significantly sensitized. Moreover, using small molecules QNZ and cabozantinib to target NFKB1 and MET, respectively, this together with lenvatinib could synergistically induce apoptosis and suppress HCC growth in vitro and in vivo. CONCLUSION: Our results demonstrated that genome-wide CRISPR/Cas9 screening is a powerful tool for the design of rational combinational cancer therapy and provided candidate genes possible for combined treatments with lenvatinib to improve therapy efficacy.

Carcinoma, Hepatocellular↗