SEARCH · PubMed Health

Results for “CRISPR/Cas9 screening”

Explore indexed PubMed citations for clinical trials, systematic reviews and public health research. Read source abstracts and follow each citation to its original PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 19 recordsLinked to original sources

PubMed2025

CRISPR/Cas9 screenings reveal the role of STX1A and CDK1 in Cathepsin G entering and killing colorectal cancer cells.

Neutrophils are the major populations of white blood cells and have been reported to facilitate cancer metastasis. Meanwhile, emerging evidence has recently suggested the anti-cancer role of neutrophils. Our previous study revealed that CB-839 and 5-FU-treated colorectal cancer (CRC) tumors recruited neutrophils and induced neutrophil extracellular traps (NETs). Cathepsin G (CTSG), which is released during NET formation, enters CRC cells through the receptor for advanced glycation end products (RAGE) and cleaves 14-3-3ε to promote apoptosis. However, the detailed mechanism underlying CTSG's anti-tumor function remains less studied. In this study, we report that CTSG enters CRC cells through RAGE-mediated endocytosis. Knocking out RAGE or inhibiting endocytosis blocks CTSG from entering CRC cells and attenuates CTSG-induced apoptosis. Furthermore, the clathrin coat assembly complex and SNARE proteins were enriched in an arrayed CRISPR/Cas9 screening targeting human membrane trafficking genes. Knocking out SNARE protein STX1A prevents the spread of CTSG in CRC cells and the induction of cleaved PARP. A pooled genome-wide CRISPR/Cas9 screening further identifies the role of CDK1 in the NET-induced killing of CRC cells. Inhibiting CDK1 protected CRC cells from killing by CTSG. Our study reveals novel mechanisms by which CTSG enters and kills CRC cells.

CDK1↗

PubMed2026

CRISPR/Cas9 screening revealed BIRC6-AS1/BIRC6 mediates abiraterone resistance via NHEJ pathway-dependent A20 degradation in prostate cancer.

Abiraterone acetate is a standard-of-care therapy for prostate cancer (PCa). However, resistance frequently emerges, often characterized by the progression to AR-independent phenotypes. Employing a genome-wide CRISPR/Cas9 library screening strategy, we identified 523 long non-coding RNAs (lncRNAs) and 2,183 protein-coding genes as potential candidates associated with abiraterone resistance. Notably, a pair of sense-antisense genes, BIRC6-AS1/BIRC6, was identified as a significant contributor to abiraterone resistance, serving as a critical survival factor in AR-independent contexts. BIRC6-AS1 depletion led to a reduction in both the mRNA and protein levels of BIRC6. Moreover, depletion of either BIRC6-AS1 or BIRC6 enhanced the sensitivity of PCa cells to abiraterone in both in vitro and in vivo settings. Further investigation revealed that BIRC6-AS1 stabilized the mRNA of BIRC6 through interaction with ILF2. Suppression of either BIRC6-AS1 or BIRC6 attenuated non-homologous end joining (NHEJ) repair activity, resulting in the disassembly of 53BP1 foci at DNA damage sites and an increased accumulation of DNA damage, thereby exposing a vulnerability in AR-independent resistant cells. Mechanistically, BIRC6 interacted with A20 and facilitated the K48-linked ubiquitination and subsequent degradation of A20 at the K337 residue. Additionally, A20 knockdown effectively reversed the abiraterone sensitivity induced by BIRC6-AS1 depletion. Collectively, our study provides a landscape of lncRNAs and protein-coding genes associated with abiraterone resistance and suggests that targeting the BIRC6-AS1/BIRC6 axis represents a potential therapeutic strategy to eradicate AR-independent resistant tumors in prostate cancer.

Journal Article↗

PubMed2026

VHL synthetic lethality screens uncover CBF-β as a negative regulator of STING.

Clear cell renal cell carcinoma (ccRCC) represents the most common form of kidney cancer and is typified by biallelic inactivation of the von Hippel-Lindau (VHL) tumour suppressor gene. Here, we undertake genome-wide CRISPR/Cas9 screening to reveal synthetic lethal interactors of VHL, and uncover that loss of Core Binding Factor β (CBF-β) causes cell death in VHL-null ccRCC cell lines and impairs tumour establishment and growth in vivo. This synthetic relationship is independent of the elevated activity of hypoxia inducible factors (HIFs) in VHL-null cells, but does involve the RUNX transcription factors that are known binding partners of CBF-β. Mechanistically, CBF-β loss leads to upregulation of type I interferon signalling, and we uncover a direct inhibitory role for CBF-β at the STING locus controlling Interferon Stimulated Gene expression. Targeting CBF-β in kidney cancer both selectively induces tumour cell lethality and promotes activation of type I interferon signalling.

Humans↗

PubMed2026

A transcription factor-focused CRISPR screen identifies SKI as a BCL11A-independent repressor of ζ-globin.

The regulation of α-like globin genes, particularly the embryonic ζ-globin gene (HBZ), remains incompletely understood. To identify transcriptional regulators of HBZ, we establish a GFP reporter system based on the HBZ-P2A-GFP allele in erythroid cell lines and conduct a CRISPR/Cas9 screen targeting 1639 transcription factors. This screen identifies SKI as a potent HBZ repressor. Functional validation shows that SKI loss increases HBZ expression without impairing erythropoiesis, whereas SKI overexpression suppresses HBZ. Tet-on-inducible SKI overexpression and auxin-inducible SKI degradation indicate that SKI rapidly represses HBZ transcription. Transcriptome profiling further reveals that SKI deletion activates HBZ while minimally affecting other erythroid genes. Mechanistically, genome-wide occupancy analyses show that SKI binds the distal enhancers HS-10 and HS-40, with partial co-occupancy by BCL11A. Despite this overlap, dual knockout of SKI and BCL11A synergistically increases HBZ expression, as does base editing of the SKI-binding site within HS-10. We also identify a naturally occurring variant (chr16:193207G>A) within this enhancer in α-thalassemia patients with elevated ζ-globin levels. Together, these findings establish SKI as a direct, BCL11A-independent transcriptional repressor of ζ-globin. This work advances our understanding of globin gene regulation and suggests targeted ζ-globin reactivation as a potential therapeutic strategy for α-thalassemia.

Enhancer↗

PubMed2026

Integrative chemical genetics platform identifies condensate modulators linked to neurological disorders.

Dysregulation of biomolecular condensates is implicated across multiple neurological disorders. However, approaches to systematically identify their modulators remain limited. Here, we expand the utility of MLF2 as a versatile condensate biomarker and develop CondenScreen, an integrated high-content screening and bioinformatics pipeline enabling identification of condensate modulators across chemical and genetic space. Screening 1760 bioactive compounds in a cellular DYT1 dystonia model, we validate the platform for condensate-targeted drug discovery, identifying drugs that prevent the accumulation of the MLF2 reporter into nuclear envelope condensates. In parallel, a genome-wide CRISPR/Cas9 screen correlates nuclear condensate abundance with genes implicated in microcephaly and over eight additional neurodevelopmental disorders. Machine learning and confocal imaging resolve distinct condensate phenotypes, with RNF26 deletion provoking nuclear envelope condensates that phenocopy hallmarks of torsin deficiency. Our study provides a scalable platform for identifying modulators of condensates and establishes a correlative connection between nuclear condensate accumulation and genes implicated in neurodevelopmental disorders.

Humans↗

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↗

PubMed2025

Suppression of OTUD4 protects against myocardial ischemia-reperfusion injury by increasing autophagic flux and inhibiting apoptosis in cardiomyocytes.

Dysregulated autophagic flux plays a critical role in myocardial ischemia-reperfusion injury (MIRI), complicating cardiac reperfusion therapy. In this study, we identified OTUD4 as a potential regulator of autophagic flux in MIRI using CRISPR/Cas9 sgRNA sequencing. However, the underlying mechanism is poorly understood. The purpose of this study is to investigate the effects of OTUD4 on autophagic flux in OGD-R treated AC16 cells (IRI model in vitro) and LAD artery ligation induced myocardial ischemia-reperfusion mice (MIRI model in vivo). In the in vitro IRI cell model, OTUD4 knockdown significantly reversed impaired autophagic flux, increased mitochondrial membrane potential, and decreased LDH activity, ROS production, autophagy and apoptosis. Overexpression of OTUD4 showed the opposite result. In the in vivo MIRI model, OTUD4 knockdown also significantly decreased infarct area, improved cardiac structure and function, reduced serum BNP and LDH levels, attenuated cardiac tissue injury/fibrosis/myocardial hypertrophy, and ultimately exerted myocardial protective effects against ischemia-reperfusion injury. Importantly, OTUD4 knockdown inhibited autophagosome-associated markers (LC3II/LC3I, Beclin1, ATG9), autophagy substrate p62, increased lysosomal activity marker LAMP2, and activated the autophagy pathway (AKT/mTOR), thereby promoting the recovery of impaired autophagic flux in the MIRI model. Moreover, OTUD4 showed strong interaction with UBAC1, and OTUD4 deficiency decreases UBAC1 protein expression by impairing its deubiquitination, thereby regulating autophagy. In short, blocking OTUD4 restored damaged autophagic flux in I/R induced myocardial injury both in vivo and in vitro, inhibited myocardial cell apoptosis, and greatly improved cardiac function in ischemia-reperfusion mice. KEY MESSAGES: OTUD4 was identified as a key negative regulator of autophagy flux in myocardial ischemia-reperfusion injury (MIRI) via genome-wide CRISPR/Cas9 screening. OTUD4 knockdown exerts cardioprotective effects by reducing apoptosis and ROS generation and improving heart function in both in vitro and in vivo models. The interaction between OTUD4 and UBAC1 was confirmed, and OTUD4 maintains UBAC1 stability through deubiquitination, providing new insights into the ubiquitination regulatory mechanism in myocardial injury. Targeting OTUD4 has therapeutic potential for MIRI, as OTUD4 knockdown alleviated MIRI in both in vitro and in vivo models, suggesting the possibility of developing OTUD4 inhibitors for cardiac reperfusion treatment.

Animals↗

PubMed2025

Integrative multiomic approaches reveal ZMAT3 and p21 as conserved hubs in the p53 tumor suppression network.

TP53, the most frequently mutated gene in human cancer, encodes a transcriptional activator that induces myriad downstream target genes. Despite the importance of p53 in tumor suppression, the specific p53 target genes important for tumor suppression remain unclear. Recent studies have identified the p53-inducible gene Zmat3 as a critical effector of tumor suppression, but many questions remain regarding its p53-dependence, activity across contexts, and mechanism of tumor suppression alone and in cooperation with other p53-inducible genes. To address these questions, we used Tuba-seqUltra somatic genome editing and tumor barcoding in a mouse lung adenocarcinoma model, combinatorial in vivo CRISPR/Cas9 screens, meta-analyses of gene expression and Cancer Dependency Map data, and integrative RNA-sequencing and shotgun proteomic analyses. We established Zmat3 as a core component of p53-mediated tumor suppression and identified Cdkn1a as the most potent cooperating p53-induced gene in tumor suppression. We discovered that ZMAT3/CDKN1A serve as near-universal effectors of p53-mediated tumor suppression that regulate cell division, migration, and extracellular matrix organization. Accordingly, combined Zmat3-Cdkn1a inactivation dramatically enhanced cell proliferation and migration compared to controls, akin to p53 inactivation. Together, our findings place ZMAT3 and CDKN1A as hubs of a p53-induced gene program that opposes tumorigenesis across various cellular and genetic contexts.

Animals↗

PubMed2026

Redox cycling of viral RNA polymerase controls picornavirus replication.

Picornaviruses, including foot-and-mouth disease virus (FMDV), enterovirus 71 (EV71) and encephalomyocarditis virus (EMCV), are important pathogens that cause fever, herpes, and myocarditis in humans and animals. The interplay between picornaviruses and their hosts remains enigmatic. Here we perform porcine genome-wide CRISPR/Cas9 screens and identify methionine sulfoxide reductase B3 (MSRB3) as an essential factor for FMDV. MSRB3 deficiency inhibits FMDV replication. Mechanistically, MSRB3 eliminates methionine oxidation of FMDV 3D polymerase and stabilizes its expression. Further studies show that radical SAM domain-containing protein 1 (RSAD1) catalyzes methionine oxidation of FMDV 3D polymerase and promotes its aggregation and subsequent degradation through the autophagy-lysosome pathway. Importantly, RSAD1-MSRB3-mediated redox modification also affects the stability of 3D polymerases of EV71 and EMCV, and regulates their infectivity and pathogenesis both in vitro and in vivo. Collectively, this study corroborates that RSAD1-MSRB3-mediated redox cycling of 3D polymerase plays a conserved function in modulating picornavirus infection, providing insights into viral pathogenesis and broad-spectrum antiviral development.

Animals↗

PubMed2026

EIF4H and YBX1 are essential host factors for hepatitis E virus replication and pathogenesis.

Hepatitis E virus (HEV) is a leading cause of acute viral hepatitis worldwide, responsible for approximately 20 million infections annually. Despite the availability of a vaccine in China, no direct-acting antivirals are approved, and host factors required for HEV replication remain poorly defined. Here, using a genome-wide CRISPR/Cas9 knockout screen in a replicon system, we identified Eukaryotic Translation Initiation Factor 4H (EIF4H) and Y-Box Binding Protein 1 (YBX1) as essential host factors for HEV replication and pathogenesis. Knockout of either factor markedly impaired replication of HEV genotypes 1, 3, and 4, as well as HEV infection and production in hepatocellular carcinoma cells and human induced pluripotent stem cell-derived hepatocyte-like cells, while leaving SARS-CoV-2, hepatitis B virus, hepatitis C virus, and Zika virus unaffected, underscoring their HEV-specific roles. Mechanistically, EIF4H interacts with ORF1 via its methyltransferase-Y-papain-like protease region, and EIF4H deficiency alters the composition of the ORF1-associated replication complex. By contrast, YBX1 is dispensable for ORF1 translation and RNA binding but is specifically required for ORF1 proteolytic processing, a prerequisite for assembling a functional replication machinery. EIF4H knockout rats and liver-specific YBX1 knockout rats were largely resistant to rat HEV-C1 infection, showing profound reductions in viral shedding, suppressed hepatic and intestinal viral loads, and protection from liver pathology. Together, our findings establish EIF4H and YBX1 as essential host factors for HEV infection and pathogenesis and reveal potential targets for antiviral intervention.

Virus Replication↗

PubMed2026

CRISPR/Cas9 loss-of-function screen in a neuronal model of AP-4 deficiency identifies ATG9A trafficking modulators.

Biallelic loss-of-function variants in adaptor protein complex 4 (AP-4) disrupt trafficking of transmembrane proteins at the trans-Golgi network, including autophagy-related protein 9A (ATG9A), leading to childhood-onset hereditary spastic paraplegia (AP-4-HSP). AP-4-HSP is characterized by features of both a neurodevelopmental and a degenerative neurological disease. To investigate the molecular mechanisms underlying AP-4-HSP and identify potential therapeutic targets, we conducted an arrayed CRISPR/Cas9 loss-of-function screen of 8,478 genes, targeting the "druggable genome," in a human neuronal model of AP-4 deficiency. Through this phenotypic screen and subsequent experiments, key modulators of ATG9A trafficking were identified, and complementary pathway analyses provided insights into the regulatory landscape of ATG9A transport. Knockdown of ANPEP and NPM1 enhanced ATG9A availability outside the trans-Golgi network, suggesting that they regulate ATG9A localization. These findings deepen our understanding of ATG9A trafficking in the context of AP-4 deficiency and offer a framework for the development of targeted interventions for AP-4-HSP.

Humans↗

PubMed2026

Genome-wide CRISPR screen identifies RNF24 as a critical host factor for foot-and-mouth disease virus entry.

BACKGROUND: Foot-and-mouth disease virus (FMDV) causes substantial economic losses in global livestock production; however, the key host factors supporting its early infection process remain poorly characterized. METHODS: In this study, we performed an unbiased genome-wide CRISPR/Cas9 knockout screening using porcine cells to screen and identify host factors involved in FMDV infection. RESULTS: We identified that the E3 ubiquitin ligase RNF24 supports efficient FMDV entry. RNF24 depletion inhibits viral entry and replication, whereas its overexpression enhances viral infectivity. Mechanistically, RNF24 preferentially promotes K27-linked non-degradative polyubiquitination of leupaxin (LPXN) at lysine 162, driving LPXN's trafficking to the plasma membrane. At the membrane, LPXN assembles a ternary integrin-LPXN-VP1 complex that strengthens virus-receptor interactions and promotes viral adsorption and entry. Disruption of this ubiquitination event via the LPXN K162R mutation impairs complex formation and compromises viral entry. CONCLUSION: Together, our study reveals a ubiquitin-dependent RNF24-LPXN regulatory axis that supports FMDV entry, highlights the role of non-degradative ubiquitination in viral pathogenesis, and proposes this interface as a potential target for antiviral intervention.

CRISPR screening↗

PubMed2023

RNF4 and USP7 cooperate in ubiquitin-regulated steps of DNA replication.

DNA replication requires precise regulation achieved through post-translational modifications, including ubiquitination and SUMOylation. These modifications are linked by the SUMO-targeted E3 ubiquitin ligases (STUbLs). Ring finger protein 4 (RNF4), one of only two mammalian STUbLs, participates in double-strand break repair and resolving DNA-protein cross-links. However, its role in DNA replication has been poorly understood. Using CRISPR/Cas9 genetic screens, we discovered an unexpected dependency of RNF4 mutants on ubiquitin specific peptidase 7 (USP7) for survival in TP53-null retinal pigment epithelial cells. TP53-/-/RNF4-/-/USP7-/- triple knockout (TKO) cells displayed defects in DNA replication that cause genomic instability. These defects were exacerbated by the proteasome inhibitor bortezomib, which limited the nuclear ubiquitin pool. A shortage of free ubiquitin suppressed the ataxia telangiectasia and Rad3-related (ATR)-mediated checkpoint response, leading to increased cell death. In conclusion, RNF4 and USP7 work cooperatively to sustain a functional level of nuclear ubiquitin to maintain the integrity of the genome.

Animals↗

PubMed2025

COG6 is an essential host factor for influenza A virus infection.

Influenza A virus (IAV) relies on the host cellular machinery to support its replication. Understanding these host dependencies can inform the development of novel antiviral strategies. In this study, we identified conserved oligomeric Golgi complex subunit 6 (COG6) as a novel host factor critical for IAV replication through a genome-wide clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) knockout screen. Disruption of COG6 significantly impaired viral replication. Mechanistically, COG6 supports IAV replication via two distinct means. First, consistent with the role of the COG complex in Golgi homeostasis, COG6 is required for the proper presentation of surface sialic acids, the primary receptor for IAV entry. Second, COG6 deficiency unexpectedly led to lysosome-dependent degradation of viral proteins. Notably, lysosomal activity was also upregulated in IAV-infected wild-type cells, albeit to a lesser extent than in COG6-deficient cells. Treatment with lysosomal inhibitors rescued viral protein stability in COG6 knockout cells. Protein interaction analysis further demonstrated that COG6-mediated stabilization of viral proteins did not rely on viral protein-COG6 interaction, refuting the hypothesis that COG6 acts as a shield factor to protect viral protein from lysosomal degradation. Moreover, knockout of other COG subunits produced similar antiviral effects, suggesting that an intact COG complex is required for IAV replication. Together, these findings uncover a critical role of the COG complex in regulating IAV replication and highlight a previously unappreciated functional link between the Golgi and lysosomes that could be exploited for treating IAV infections.IMPORTANCEDespite advances in virology, numerous host determinants facilitating influenza A virus (IAV) pathogenesis remain uncharacterized. Our study establishes conserved oligomeric Golgi complex subunit 6 (COG6) as a critical host factor promoting IAV infection through complementary mechanisms: receptor modulation and viral protein stabilization. This represents the first demonstration that the COG complex regulates viral pathogenesis through proteostasis mechanisms, fundamentally expanding our understanding of host-virus interactions at the organelle interface. These findings not only provide new perspectives on viral exploitation of Golgi trafficking networks but also identify potential therapeutic targets against evolving influenza strains.

Influenza A virus↗

PubMed2024

Mannose receptor (MRC1) mediates uptake of dextran in macrophages via receptor-mediated endocytosis.

UNLABELLED: Macrophages maintain surveillance of their environment using receptor-mediated endocytosis and pinocytosis. Receptor-mediated endocytosis allows macrophages to recognize and internalize specific ligands whereas macropinocytosis non-selectively internalizes extracellular fluids and solutes. Here, CRISPR/Cas9 whole-genome screens were used to identify genes regulating constitutive and growth factor-stimulated dextran uptake in murine bone-marrow derived macrophages (BMDM). The endocytic mannose receptor c-type 1 ( Mrc1 , also known as CD206) was a top hit in the screen. Targeted gene disruptions of Mrc1 reduced dextran uptake but had little effect on uptake of Lucifer yellow, a fluid-phase marker. Other screen hits also differentially affected the uptake of dextran and Lucifer yellow, indicating the solutes are internalized by different mechanisms. We further deduced that BMDMs take up dextran via MRC1-mediated endocytosis by showing that competition with mannan, a ligand of MRC1, as well as treatment with Dyngo-4a, a dynamin inhibitor, reduced dextran uptake. Finally, we observed that IL4-treated BMDM internalize more dextran than untreated BMDM by upregulating MRC1 expression. These results demonstrate that dextran is not an effective marker for the bulk uptake of fluids and solutes by macropinocytosis since it is internalized by both macropinocytosis and receptor-mediated endocytosis in cells expressing MRC1. This report identifies numerous genes that regulate dextran internalization in primary murine macrophages and predicts cellular pathways and processes regulating MRC1. This work lays the groundwork for identifying specific genes and regulatory networks that regulate MRC1 expression and MRC1-mediated endocytosis in macrophages. SIGNIFICANCE STATEMENT: Macrophages constantly survey and clear tissues by specifically and non-specifically internalizing debris and solutes. However, the molecular mechanisms and modes of regulation of these endocytic and macropinocytic processes are not well understood. Here, CRISPR/Cas9 whole genome screens were used to identify genes regulating uptake of dextran, a sugar polymer that is frequently used as a marker macropinocytosis, and compared with Lucifer yellow, a fluorescent dye with no known receptors. The authors identified the mannose receptor as well as other proteins regulating expression of the mannose receptor as top hits in the screen. Targeted disruption of Mrc1 , the gene that encodes mannose receptor, greatly diminished dextran uptake but had no effect on cellular uptake of Lucifer yellow. Furthermore, exposure to the cytokine IL4 upregulated mannose receptor expression on the cell surface and increased uptake of dextran with little effect on Lucifer yellow uptake. Studies seeking to understand regulation of macropinocytosis in macrophages will be confounded by the use of dextran as a fluid-phase marker. MRC1 is a marker of alternatively activated/anti-inflammatory macrophages and is a potential target for delivery of therapeutics to macrophages. This work provides the basis for mechanistic underpinning of how MRC1 contributes to the receptor-mediated uptake of carbohydrates and glycoproteins from the tissue milieu and distinguishes genes regulating receptor-mediated endocytosis from those regulating the bona fide fluid-phase uptake of fluids and solutes by macropinocytosis.

Journal Article↗

PubMed2026

PRMT3 restricts porcine epidemic diarrhea virus replication by disrupting the interaction between VAPA and the viral nucleocapsid protein.

Porcine epidemic diarrhea virus (PEDV) represents a severe threat to the global swine industry. Its infection process involves intricate virus-host interactions and immune evasion mechanisms, but effective therapeutic targets remain elusive. In this study, we identified protein arginine methyltransferase 3 (PRMT3) as a novel regulatory factor that significantly modulates PEDV infection via genome-wide CRISPR/Cas9 knockout library screening. Knockout or inhibition of PRMT3 markedly enhanced PEDV infection in multiple cell lines, including LLC-PK1, IPEC-J2, and primary porcine intestinal epithelial cells. Mechanistic investigations revealed that PRMT3 can restrict PEDV infection by interacting with vesicle-associated membrane protein-associated protein A (VAPA). Further analysis revealed that VAPA facilitates cholesterol transport through binding to oxysterol-binding protein (OSBP) and inhibits the autophagic degradation of the viral nucleocapsid (N) protein, with both processes being critical for promoting PEDV infection in host cells. A detailed analysis revealed that K52 within its major sperm protein (MSP) domain interacts with D404 and D405 in the two phenylalanines in an acidic tract (FFAT)-like motifs of the N protein, and these interactions proved essential for PEDV infection. In summary, this is the first study to identify and validate the PRMT3-VAPA-N protein autophagic degradation axis as a key pathway through which PRMT3 suppresses PEDV infection, with VAPA acting as an essential host factor for PEDV pathogenesis. These findings uncover novel signaling pathways and molecular targets for the development of anti-PEDV therapeutics.

Animals↗

PubMed2024

Mannose receptor (MRC1) mediates uptake of dextran by bone marrow-derived macrophages.

Macrophages survey their environment using receptor-mediated endocytosis and pinocytosis. Receptor-mediated endocytosis allows internalization of specific ligands, whereas pinocytosis nonselectively internalizes extracellular fluids and solutes. CRISPR/Cas9 whole-genome screens were used to identify genes regulating constitutive and growth factor-stimulated dextran uptake in murine bone marrow-derived macrophages (BMDM). The mannose receptor c-type 1 (MRC1/CD206) was a top hit in the screen. Targeted gene disruptions of Mrc1 reduced dextran uptake but had little effect on fluid-phase uptake of Lucifer yellow. Other screen hits also differentially affected the uptake of dextran and Lucifer yellow, indicating internalization by separate mechanisms. Visualization of dextran and Lucifer yellow uptake by microscopy showed enrichment of dextran in small puncta, which was inhibitable by mannan, a ligand of MRC1. In contrast, Lucifer yellow predominantly was internalized in larger macropinosomes. In addition, IL4-treated BMDMs internalized more dextran than untreated BMDM correlating with increased MRC1 expression. Therefore, dextran is not an effective marker for pinocytosis in BMDMs since it is internalized by receptor-mediated process. Numerous genes that regulate dextran internalization in primary murine macrophages were identified in the whole-genome screens, which can inform understanding of the regulation of MRC1 expression and MRC1-mediated uptake in macrophages.

Animals↗

PubMed2026

In vivo CRISPR screening identifies metastasis suppressors in triple-negative breast cancer.

Metastatic cancer remains the leading cause of cancer-related mortality, yet tumor cell-intrinsic mechanisms restraining metastatic dissemination remain incompletely defined. Here, we perform an unbiased in vivo genome-wide CRISPR/Cas9 loss-of-function screen in a breast cancer xenograft model to identify regulators of metastatic progression. This approach uncovers clinically relevant metastasis suppressor genes (MSGs), including VPS45, CMTR2, RBSN, and NF2, whose loss enhances lung colonization. Functional validation demonstrates that depletion of these genes promotes epithelial-to-mesenchymal transition, migration, invasion, intravasation, and angiogenesis, whereas CRISPR-mediated activation suppresses metastatic spread. Integration with patient datasets reveals reduced expression in tumors and associations with advanced disease, with higher expression trending toward improved outcomes. Notably, CMTR2 loss induces vascular remodeling and intratumoral heterogeneity, supporting a role in tumor-vascular interactions. Collectively, this study identifies a network of MSGs that constrain tumor dissemination and highlights the power of in vivo CRISPR functional genomics to uncover regulators of metastatic disease.

Humans↗