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Results for “CRISPR knockout”

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PubMed2026

A CRISPR-Cas9 screen identifies LAPTM4A (lysosomal protein transmembrane 4 alpha) as a key host barrier against PRRSV infection.

Porcine reproductive and respiratory syndrome virus (PRRSV) manipulates host intracellular processes, particularly macroautophagy/autophagy and lysosomal function, to facilitate its replication and spread. However, the precise host factors and molecular mechanisms by which PRRSV remodels the autophagy-lysosome axis remain poorly defined. Here, we performed a CRISPR-Cas9 knockout screen targeting 1,332 genes involved in protein degradation, metabolism, and vesicular trafficking, and identified LAPTM4A (lysosomal protein transmembrane 4 alpha) as a critical antiviral factor involved in the lysosomal pathway. A yeast two-hybrid screen identified LAPTM4A as an interactor of PRRSV GP5 (glycoprotein 5). Mechanistically, GP5 recruits the E3 ubiquitin ligase NEDD4 and the autophagy receptor SQSTM1/p62 to promote K63-linked polyubiquitination of LAPTM4A, leading to its autophagic degradation. This selective degradation activates the AMPK-ULK1-MAP1LC3/LC3 signaling cascade, initiating autophagy while facilitating MTOR-lysosome colocalization, thereby suppressing TFEB nuclear translocation and transcription of lysosome-related genes. The resulting incomplete autophagic flux enhances viral replication. Additionally, in terms of host defense, LAPTM4A maintains lysosomal homeostasis by restraining excessive autophagy through AMPK-ULK1-LC3 signaling and promoting TFEB-dependent lysosomal gene expression by impairing the binding of RPTOR/raptor to MTOR, thus providing broad antiviral protection against multiple RNA viruses. Collectively, our findings identify LAPTM4A as a central regulator of lysosome-autophagy homeostasis and reveal a viral strategy that dismantles this defense axis to facilitate infection.Abbreviations: ATG5: autophagy related 5; AMPK: adenosine 5'-monophosphate (AMP)-activated protein kinase; Baf A1: bafilomycin A1; CHX: cycloheximide; Co-IP: co-immunoprecipitation; DMVT library: protein degradation, metabolism, and vesicular trafficking library; LAPTM4A: lysosomal protein transmembrane 4 alpha; MAGeCK: model-based analysis of genome-wide CRISPR-Cas9 knockout; MOI: multiplicity of infection; MTOR: mechanistic target of rapamycin kinase; NC: negative control; PAMs: porcine alveolar macrophages; PRKAA/AMPKα: protein kinase AMP-activated catalytic subunit alpha; PRRSV: porcine reproductive and respiratory syndrome virus; qRT-PCR: quantitative real-time PCR; siRNA: small interfering RNA; SQSTM1/p62: sequestosome 1; TCID50: 50% tissue culture infective dose; TFEB: transcription factor EB; Ub: ubiquitin; ULK1: unc-51 like autophagy activating kinase 1; WT: wild type.

Animals↗

PubMed2026

An SLC7A5-dependent nutrient-sensing circuit overcomes cisplatin tolerance via mTOR-autophagy signaling.

Cisplatin-based chemotherapy responses are highly heterogeneous across cancers, with the mechanisms governing drug sensitivity remaining incompletely understood. Using genome-wide CRISPR-Cas9 knockout screening, we systematically characterized regulators of cisplatin response and uncovered a counterintuitive finding: mTOR inhibition promotes cisplatin tolerance, contradicting the canonical view that PI3K-AKT-mTOR activation confers chemoresistance. Mechanistically, both mTOR suppression and cisplatin treatment converge to activate cytoprotective autophagy, which enhances cancer cell survival under therapeutic stress. The amino acid transporter SLC7A5 was identified and validated as a key integrator of the mTOR-autophagy axis that modulates cisplatin sensitivity. SLC7A5 expression positively correlates with cisplatin sensitivity across cancer cell lines, and its downregulation is associated with cisplatin resistance in multiple cancer types, supporting its potential as a mechanistically grounded predictive biomarker. Translationally, leucine supplementation sensitizes cancer cells to cisplatin in an SLC7A5-mTOR-autophagy-dependent manner. Collectively, our study defines a novel mTOR-autophagy adaptive loop governing cisplatin tolerance, positions SLC7A5 as a central regulatory node with both biomarker and therapeutic target value, and proposes leucine supplementation as a simple, translatable strategy to improve cisplatin efficacy in SLC7A5-expressing tumors.

Cisplatin↗

PubMed2026

Pcgf5 controls the exit from totipotency in mouse embryonic stem cells.

Mouse embryonic stem cell (ESC) cultures contain a rare subpopulation of two-cell-like cells (2CLCs) that transiently reactivate a two-cell embryo-like transcriptional program characteristic of zygotic genome activation (ZGA), including the endogenous retrovirus MERVL and Zscan4, and thereby regain a totipotent-like state. Polycomb repressive complex 1 (PRC1)-mediated H2AK119ub1 has been implicated in restraining entry into the 2C-like state through Pcgf6, yet the factors governing exit from this state and loss of totipotency remain poorly defined. Here, we show that among the six Pcgf paralogs, Pcgf5, which is most prominently upregulated in 2CLCs and forms an MERVL-driven chimeric transcript (Pcgf5MT2C_Mm) during ZGA in 2-cell embryos, controls exit from the 2C-like state in mouse ESCs. Using a reporter ESC line carrying MERVL-tdTomato and Zscan4c-EGFP (MtZG), we manipulated Pcgf5 dosage bidirectionally. Doxycycline (Dox)-inducible overexpression (OE) of Pcgf5 reduced the double-positive (DP) 2C-like population. Conversely, CRISPR-mediated knockout (KO) of Pcgf5 by targeting a common exon shared by all Pcgf5 variants (hereafter, total Pcgf5) increased the DP population. Time-lapse imaging directly confirmed that these changes reflected genuine differences in duration of the 2C-like state: OE shortened, whereas KO prolonged, the time cells spent in this state. These findings reveal that a Polycomb group factor controls not only entry into but also exit from the 2C-like state.

Animals↗

PubMed2024

Myoferlin: A Potential Marker of Response to Radiation Therapy and Survival in Locally Advanced Rectal Cancer.

PURPOSE: Patients with locally advanced rectal cancer often require neoadjuvant chemoradiation therapy to downstage the disease, but the response is variable with no predictive biomarkers. We have previously revealed through proteomic profiling that myoferlin is associated with response to radiation therapy. The aims of this study were to further validate this finding and explore the potential for myoferlin to act as a prognostic and/or therapeutic target. METHODS AND MATERIALS: Immunohistochemical analysis of a tissue microarray (TMA) for 111 patients was used to validate the initial proteomic findings. Manipulation of myoferlin was achieved using small interfering RNA, a small molecular inhibitor (wj460), and a CRISPR-Cas9 knockout cell line. Radiosensitization after treatment was assessed using 2-dimensional clonogenic assays, 3-dimensional spheroid models, and patient-derived organoids. Underlying mechanisms were investigated using electrophoresis, immunofluorescence, and immunoblotting. RESULTS: Analysis of both the diagnostic biopsy and tumor resection samples confirmed that low myoferlin expression correlated with a good response to neoadjuvant long-course chemoradiation therapy. High myoferlin expression was associated with spread to local lymph nodes and worse 5-year survival (P = .01; hazard ratio, 3.5; 95% CI, 1.27-10.04). This was externally validated using the Stratification in Colorectal Cancer database. Quantification of myoferlin using immunoblotting in immortalized colorectal cancer cell lines and organoids demonstrated that high myoferlin expression was associated with increased radioresistance. Biological and pharmacologic manipulation of myoferlin resulted in significantly increased radiosensitivity across all cell lines in 2-dimensional and 3-dimensional models. After irradiation, myoferlin knockdown cells had a significantly impaired ability to repair DNA double-strand breaks. This appeared to be mediated via nonhomologous end-joining. CONCLUSIONS: We have confirmed that high expression of myoferlin in rectal cancer is associated with poor response to neoadjuvant therapy and worse long-term survival. Furthermore, the manipulation of myoferlin led to increased radiosensitivity in vitro. This suggests that myoferlin could be targeted to enhance the sensitivity of patients with rectal cancer to radiation therapy, and further work is required.

Humans↗

PubMed2026

Genome-scale CRISPR screening uncovers SRSF6 as a target to sensitize hepatocellular carcinoma to radiotherapy.

BACKGROUND & AIMS: Radiotherapy confers clinical benefits to patients with hepatocellular carcinoma (HCC) across all stages, yet its clinical efficacy is limited by radioresistance. This study aimed to identify key regulators of HCC radiosensitivity through genome-wide functional screening. METHODS: A genome-wide CRISPR-Cas9 screen in Huh7 cells identified radiosensitivity regulators, with SRSF6 validated by siRNA knockdown and γ-H2AX assessment. Stable shRNA-mediated SRSF6 knockdown was established in Huh7 and HepG2 cells, followed by clonogenic, EdU incorporation, apoptosis, micronucleus, and comet assays. Mechanistically, RNA-seq, Western blotting, mRNA stability assays, RIP-qPCR, and RAD51 overexpression rescue assays were performed. The therapeutic potential of the SRSF6 inhibitor indacaterol was evaluated using MTS assays, HCC xenograft mouse models (BALB/c-nu/nu, n = 28), and HCC patient-derived organoids (PDOs) (n = 3). In addition, SRSF6 expression and its correlation with patient survival were analyzed using data from The Cancer Genome Atlas and a tissue microarray (n = 14 HCC and 14 paired adjacent non-tumorous liver samples). RESULTS: We identified the RNA-binding protein SRSF6 as a driver of HCC radioresistance. SRSF6 depletion enhanced the radiosensitivity of HCC cells (p <0.05-0.0001) by post-transcriptionally destabilizing the mRNAs of critical DNA repair genes (p <0.05-0.0001), thereby impairing radiation-induced DNA damage repair. The radiosensitizing effect of SRSF6 depletion was partially abrogated by ectopic overexpression of the core DNA repair protein RAD51 (p <0.05-0.001). Indacaterol exhibited cytotoxic effects on HCC cells (p <0.05-0.0001) and enhanced the antitumor efficacy of radiation in vivo (p <0.05-0.0001), as further validated across multiple HCC patient-derived organoids (p <0.05-0.0001). CONCLUSIONS: SRSF6 is a key regulator of HCC radioresistance through its post-transcriptional control of DNA repair capacity, and represents a novel therapeutic target to sensitize HCC to radiotherapy. IMPACT AND IMPLICATIONS: In this study, we performed a genome-wide CRISPR-Cas9 knockout library screen to dissect the molecular determinants governing HCC radiosensitivity, and identified RNA-binding protein SRSF6 as a driver of HCC radioresistance. We demonstrate that SRSF6 depletion disrupts the post-transcriptional stability of key DNA repair gene mRNAs and enhances HCC radiosensitivity. These findings are important for radiation oncologists and translational researchers, as they identify SRSF6-dependent RNA regulation as a critical determinant of radiotherapy response in HCC. Practically, we show that the clinically approved bronchodilator indacaterol suppresses SRSF6 function and enhances the antitumor efficacy of radiotherapy, offering a readily repurposable pharmacological strategy to overcome radioresistance. These implications are based on preclinical evidence across multiple models; however, future clinical trials are needed to validate the safety and efficacy of indacaterol-based radiosensitization in patients with HCC.

DNA repair↗

PubMed2024

UDP-glycosyltransferases act as key determinants of host plant range in generalist and specialist Spodoptera species.

Phytophagous insects have evolved sophisticated detoxification systems to overcome the antiherbivore chemical defenses produced by many plants. However, how these biotransformation systems differ in generalist and specialist insect species and their role in determining insect host plant range remains an open question. Here, we show that UDP-glucosyltransferases (UGTs) play a key role in determining the host range of insect species within the Spodoptera genus. Comparative genomic analyses of Spodoptera species that differ in host plant breadth identified a relatively conserved number of UGT genes in generalist species but high levels of UGT gene pseudogenization in the specialist Spodoptera picta. CRISPR-Cas9 knockouts of the three main UGT gene clusters of Spodoptera frugiperda revealed that UGT33 genes play an important role in allowing this species to utilize the poaceous plants maize, wheat, and rice, while UGT40 genes facilitate utilization of cotton. Further functional analyses in vivo and in vitro identified the UGT SfUGT33F32 as the key mechanism that allows generalist S. frugiperda to detoxify the benzoxazinoid DIMBOA (2,4-dihydroxy-7-methoxy-2H-1,4-benzoxazin-3(4H)-one), a potent insecticidal phytotoxin produced by poaceous plants. However, while this detoxification capacity is conserved in several generalist Spodoptera species, Spodoptera picta, which specializes on Crinum plants, is unable to detoxify DIMBOA due to a nonfunctionalizing mutation in SpUGT33F34. Collectively, these findings provide insight into the role of insect UGTs in host plant adaptation, the mechanistic basis of evolutionary transitions between generalism and specialism and offer molecular targets for controlling a group of notorious insect pests.

Animals↗

PubMed2025

CROPseq-multi: a universal solution for multiplexed perturbation in high-content pooled CRISPR screens.

Forward genetic screens seek to dissect complex biological systems by systematically perturbing genetic elements and observing the resulting phenotypes. While standard screening methodologies introduce individual perturbations, multiplexing perturbations improves the performance of single-target screens and enables combinatorial screens for the study of genetic interactions. Current tools for multiplexing perturbations are limited by technical challenges and do not offer compatibility across diverse screening methodologies, including enrichment, single-cell sequencing, and optical pooled screens. Here, we report the development of CROPseq-multi (CSM), a CROPseq1-inspired lentiviral system to multiplex Streptococcus pyogenes (Sp) Cas9-based perturbations with versatile readout compatibility and high performance for both perturbation and barcode identification. CSM has equivalent per-guide activity to CROPseq and low lentiviral recombination frequencies. Dual-guide CSM libraries are constructed in a single, facile molecular cloning step that facilitates the use of unique molecular identifiers. CSM is compatible with enrichment screening methodologies, single-cell RNA-sequencing readouts, and optical pooled screens. For optical pooled screens, an optimized and multiplexed in situ detection protocol improves barcode counts 10-fold (for mRNA detection), enables detection of recombination events, and reduces the number of sequencing cycles required for decoding by 3-fold relative to CROPseq. CROPseq-multi-v2 (CSMv2) adds compatibility for detection methods based on T7 RNA polymerase in vitro transcription2-5. CSM provides a single system for CRISPR screens that is compatible with individual and combinatorial perturbations, diverse SpCas9-based perturbation technologies, and multiple high-content, single-cell phenotypic readouts.

CRISPR Cas9↗

PubMed2025

Genome reorganisation and expansion shape 3D genome architecture and define a distinct regulatory landscape in coleoid cephalopods.

How genomic changes translate into organismal novelties is often confounded by the multi-layered nature of genome architecture and the long evolutionary timescales over which molecular changes accumulate. Coleoid cephalopods (squid, cuttlefish, and octopus) provide a unique system to study these processes due to a large-scale chromosomal rearrangement in the coleoid ancestor that resulted in highly modified karyotypes, followed by lineage-specific fusions, translocations, and repeat expansions. How these events have shaped gene regulatory patterns underlying the evolution of coleoid innovations, including their large and elaborately structured nervous systems, novel organs, and complex behaviours, remains poorly understood. To address this, we integrate Micro-C, RNA-seq, and ATAC-seq across multiple coleoid species, developmental stages, and tissues. We find that while topological compartments are broadly conserved, hundreds of chromatin loops are species- and context-specific, with distinct regulation signatures and dynamic expression profiles. CRISPR-Cas9 knockout of a putative regulatory sequence within a conserved region demonstrates the role of loops in neural development and the prevalence of long-range, inter-compartmental interactions. We propose that differential evolutionary constraints across the coleoid 3D genome allow macroevolutionary processes to shape genome topology in distinct ways, facilitating the emergence of novel regulatory entanglements and ultimately contributing to the evolution and maintenance of complex traits in coleoids.

Journal Article↗

PubMed2026

Genome-wide association studies of plant traits and functional analysis of leaf development-related genes in citrus.

Labor-saving and high-light-efficiency tree architecture is a key breeding objective for woody fruit trees like citrus. However, population genetics information on these traits remains limited. In this study, tree architecture, thorn, and leaf traits were evaluated in 353 F2 progeny derived from a cross between Clementine mandarin and precocious trifoliate orange-an early-flowering variety. A random subset of 300 offspring was sequenced for a genome-wide association study (GWAS), which detected 10 216 significantly associated SNPs and defined several major quantitative trait loci (QTLs) for the target traits. Subsequent bulked segregant analysis (BSA) and GWAS on individuals with extreme compound leaf phenotypes mapped the causal gene(s) to a 0.8 Mb region (22.15-22.95 Mb) on chromosome 4. Genetic analysis across multiple hybrid combinations confirmed that the compound leaf trait in trifoliate orange is dominantly inherited and follows Mendelian segregation. Transcriptome profiling of parental leaves at different developmental stages identified a KNOX gene, CiKNAT6, as a candidate. Further validation using CAPS markers and Hi-Tom sequencing demonstrated tight linkage between an InDel polymorphism in CiKNAT6 and leaf shape across diverse citrus species and the F2 population, with co-segregation observed for the compound leaf trait. Due to alternative splicing producing seven splice variants, the CiKNAT6 DNA sequence was selected for genetic transformation experiments. Functional analysis revealed that the Clementine mandarin allele of CiKNAT6 is non-functional owing to an InDel, whereas ectopic expression of the trifoliate orange allele in tobacco and lemon induced leaf curling and reduced leaf size. CRISPR-Cas9 knockout of CiKNAT6 in trifoliate orange resulted in increased leaf area. These findings provide valuable genetic resources and insights for future studies on tree architecture and leaf morphology.

Plant Leaves↗

PubMed2026

Rab10 coordinates SADS-CoV non-lytic egress through the ERGIC-TGN-lysosome trafficking pathway.

Swine acute diarrhea syndrome coronavirus (SADS-CoV) is a bat-originated alphacoronavirus that causes devastating enteric disease in neonatal piglets and possesses significant potential for cross-species transmission. While the early stages of the coronavirus life cycle have been extensively characterized, the host factors indispensable for virion assembly and subsequent export remain largely enigmatic. Here, by performing a genome-wide CRISPR-Cas9 knockout screen using a recombinant icSADS-CoV-GFP reporter virus, we identified the small GTPase Rab10 as a critical host dependency factor for SADS-CoV infection. Viral life cycle analysis revealed that Rab10 is not required for viral attachment, entry, or initial genome replication, but is essential for the virion transport and non-lytic egress. Rab10 deficiency markedly reduced the extracellular release of viral RNA, viral proteins, and infectious progeny, as well as the secretion of SADS-CoV virus-like particles. Confocal imaging showed that Rab10 and viral protein-positive intracellular structures were associated with LMAN1, TGN46, and LAMP1 positive compartments. These findings support a model in which Rab10 coordinates a virus-containing vesicles trafficking pathway associated with ERGIC-TGN-lysosome compartments. Mechanistically, Rab10 facilitates the loading of the viral envelope (E) protein into transport vesicles derived from the ERGIC. Rab10 associates with the SADS-CoV E protein, and mapping analyses implicated the C-terminal PDZ-binding motif, particularly residue V75, in efficient Rab10 association and viral release. Collectively, our findings identify Rab10 as a host regulator of SADS-CoV non-lytic egress and highlight the E-Rab10 interaction and the vesicular trafficking machinery as a potential target for developing antiviral strategies.

Animals↗

PubMed2025

Low-temperature embryo incubation suppresses off-target mutagenesis during CRISPR-Cas9 genome editing in medaka (Oryzias latipes) and zebrafish (Danio rerio).

Gene knockout using CRISPR-Cas9 is often employed in research aimed at elucidating gene functions in fish. However, CRISPR-Cas9 sometimes introduces unintended alterations, known as off-target mutations. These mutations can reduce the robustness of data during phenotypic analysis. In this study, we focused on the culture temperature, which is known to significantly influence mutagenesis, and examined whether low-temperature culture after introducing CRISPR-Cas9 into early embryos of medaka and zebrafish suppresses off-target mutations. Continuous incubation of medaka at 16 °C significantly reduced off-target mutation rates compared to those at 28 °C; the drawback is that it decreased the survival rate of medaka embryos. Therefore, low-temperature incubation was limited to early development in both zebrafish and medaka, and then the temperature was increased to 28 °C. Under these conditions, the mutation rates of the three off-target regions in medaka (Off-D, Off-P, and Off-A) significantly decreased, whereas those of the three target regions (DJ-1, p4hb, and avt) were unaffected. Similarly, the mutation rate of the zebrafish target region (ywhaqa) remained high, whereas the off-target (Off-Y1) mutation rate significantly reduced. Furthermore, this method effectively suppressed the germ line transmission of off-target mutations in medaka. This approach is effective to obtain more reliable data from the G0 generation of medaka and zebrafish and may reduce the screening effort required to remove individuals with off-target mutations in the F1 generation.

Animals↗

PubMed2026

Multimodal Analysis Reveals Aberrant Expression of SUMO2 and Its Significant Association With Key Mechanisms of Metabolic Pathways in Hepatocellular Carcinoma.

BACKGROUND: Hepatocellular carcinoma (HCC) is the third leading cause of cancer-related deaths worldwide. However, the role of small ubiquitin-like modifier 2 (SUMO2), a core member of the small ubiquitin-like modifier (SUMO) family, regarding its expression patterns and metabolism-related functions in HCC remains inadequately understood. METHODS: A multidimensional analytical framework was applied, integrating immunohistochemistry (153 HCC vs. 21 non-HCC samples), proteomics (159 paired samples), bulk transcriptomics (3240 HCC vs. 2267 non-HCC samples), single-cell RNA sequencing (RNA-seq) (10 HCC vs. 8 non-HCC samples), spatial transcriptomics, and external CRISPR/Cas9 functional genomics data. Systematic analyses included standardized mean difference (SMD), pathway enrichment, pseudotime trajectory inference, in silico knockout, cell-cell communication, metabolic flux scoring, immune infiltration, clinical correlation, drug sensitivity prediction, and molecular docking. RESULTS: At the protein level, immunohistochemistry (nuclear positivity) and external proteomic data collectively demonstrated consistent SUMO2 overexpression in HCC. Consistent upregulation was also observed at the mRNA level across large-scale cohorts. Single-cell RNA-seq and spatial transcriptomics localized SUMO2 enrichment to malignant hepatocytes and tumor-dominant regions. CRISPR-mediated SUMO2 knockout suppressed proliferation in multiple HCC cell lines. Mechanistically, high SUMO2 expression was significantly associated with metabolic reprogramming involving glycolysis/gluconeogenesis, pyruvate metabolism, and the tricarboxylic acid cycle. SUMO2-high malignant hepatocyte subpopulations exhibited enhanced activity of the macrophage migration inhibitory factor signaling axis and enhanced iron-sensor interactions. Further, the immune infiltration analysis revealed a negative correlation between SUMO2 expression and M1 macrophages and a positive correlation between follicular helper T cells and regulatory T cells. Clinically, elevated SUMO2 levels were found to be associated with adverse prognostic features. Furthermore, high SUMO2 expression was associated with increased sensitivity to dasatinib, and molecular docking simulations predicted potential binding between SUMO2 and dasatinib, with a Vina score of -8.5 kcal/mol. CONCLUSIONS: SUMO2 is aberrantly expressed at the protein, mRNA, single-cell, and spatial transcriptomic levels in HCC and is significantly associated with metabolic reprogramming and altered migration inhibitory factor (MIF)-mediated intercellular communication, suggesting its potential as a novel biomarker for diagnosis and treatment.

Humans↗

PubMed2026

CRISPR-Cas9-induced genetic mosaicism in three species of the microcrustacean Daphnia.

Genetic mosaicism can arise from in vivo CRISPR-Cas9 gene editing, especially in the embryos. This study evaluates the extent of genetic mosaicism resulted from CRISPR-Cas9-mediated knockout for 11 genes in the freshwater microcrustacean Daphnia magna, Daphnia pulex, and Daphnia sinensis. Based on extensive genotyping data of the asexually produced progenies of successfully edited females, we find strong evidence of mosaicism in 9 of these genes. The genotyping data also suggest that the gene editing activity can take place as early as the one-cell embryo stage and extends into the 32-cell and later stages. This study establishes genetic mosaicism as an important feature of Cas9-mediated gene editing in Daphnia.

Animals↗

PubMed2026

PDE4DIP-Derived MMG8 Supports Proliferation, Migration, and Tumor Growth in Hepatocellular Carcinoma Models.

BACKGROUND: PDE4DIP encodes a scaffold protein that has been implicated in compartmentalized signaling and cytoskeletal organization, but the role of its myomegalin variant 8 (MMG8) isoform in hepatocellular carcinoma (HCC) remains unclear. To address this gap, we examined PDE4DIP expression in public HCC datasets and investigated the functional role of MMG8 in HCC models. METHODS: PDE4DIP expression was analyzed in The Cancer Genome Atlas Liver Hepatocellular Carcinoma (TCGA-LIHC) cohort and two Gene Expression Omnibus (GEO) cohorts (GSE14520, GSE36376). MMG8 function was assessed in Huh7 cells using siRNA-mediated knockdown and in Hepa1-6 cells using lentiviral Clustered Regularly Interspaced Short Palindromic Repeats - CRISPR-associated protein 9 (CRISPR-Cas9)-mediated knockout. Cell proliferation in MMG8-KD Huh7 cells and MMG8-KO Hepa1-6 cells was assessed using Cell Counting Kit-8 (CCK-8) assays, while Huh7 cell migration was evaluated using Transwell assays. Tumor growth was assessed using a murine subcutaneous tumor model. Immunohistochemical staining for Ki67 and cleaved caspase-3 was employed to assess tumor cell proliferation and apoptosis-associated changes, respectively. Gene set enrichment analysis was performed in TCGA-LIHC tumors stratified based on PDE4DIP expression. RESULTS: PDE4DIP expression differed between tumor and non-tumor tissues across HCC cohorts, although the directionality of this difference was not uniform. MMG8 knockdown in Huh7 cells reduced proliferation and migratory activity. A single-cell-derived MMG8-KO Hepa1-6 clone exhibited reduced proliferation in vitro and formed smaller tumors in vivo, with lower Ki67 positivity but no significant difference in cleaved caspase-3 positivity between groups. In tumors from the TCGA-LIHC cohort, PDE4DIP expression was associated with distinct transcriptional programs. Specifically, PDE4DIP-high tumors presented with positive normalized enrichment score (NES) values for several metabolic pathways, whereas adhesion/extracellular matrix (ECM), cell cycle/proliferation, and translation/ribosome-related pathways exhibited negative NES values. CONCLUSIONS: These findings support a functional contribution of MMG8 to proliferative, migratory, and tumor-growth phenotypes in the tested HCC models. Bulk gene-level PDE4DIP expression in human tumors was associated with context-dependent transcriptional states and should not be interpreted as a direct surrogate for MMG8 function.

Liver Neoplasms↗

PubMed2026

Integrated multi-omics analysis and functional experiments reveals PPAP2C as a potential prognostic biomarker and therapeutic target in breast cancer.

BACKGROUND: This study aims to systematically elucidate the clinical significance and biological function of the phospholipid phosphatase (PLPP) family member (PPAP2C) phosphatidic acid phosphatase type 2C in breast cancer, and to evaluate its potential as a prognostic biomarker and therapeutic target. METHODS: Gene expression data from The Cancer Genome Atlas (TCGA), Genotype-Tissue Expression (GTEx), and Cancer Cell Line Encyclopedia (CCLE) databases were integrated to characterize the expression profile of PLPP family members, focusing on PPAP2C in breast cancer. The prognostic value of PPAP2C, initially identified at the mRNA level (TCGA, (METABRIC) Molecular Taxonomy of Breast Cancer International Consortium, Gene Expression Omnibus (GEO)), was confirmed at the protein level by immunohistochemistry (IHC) on tissue microarrays (TMA). The oncogenic functions of PPAP2C were investigated in triple-negative breast cancer (TNBC) cells through CRISPR-Cas9-mediated knockout and ectopic overexpression, with assessment of key phenotypes including proliferation, colony formation, migration, and invasion. In vivo validation was subsequently performed using an MDA-MB-231 xenograft model. RESULTS: PPAP2C exhibits the most significant overexpression pattern across 33 cancer types (upregulated in 16 cancers, downregulated in only 3). Compared with normal tissues, PPAP2C showed specific overexpression in breast cancer tissues and was significantly associated with advanced clinical stages and aggressive subtypes (HER2+ and TNBC). Survival analysis demonstrated that high PPAP2C expression correlated with significantly shorter overall survival and disease-free survival, which was further validated in METABRIC and GEO cohorts. Tissue microarray analysis confirmed higher PPAP2C protein positivity in tumor tissues (94.7%) than in adjacent normal tissues (59.7%), with worse OS and RFS in high-expression groups. Multivariate analysis identified PPAP2C as an independent prognostic factor for OS. Functional experiments revealed that PPAP2C knockout (via 5-bp/1-bp frameshift mutations) suppressed TNBC cell proliferation, colony formation, migration, and invasion, while overexpression enhanced these phenotypes. In vivo studies further demonstrated complete tumor regression in MDA-MB-231 xenografts upon PPAP2C knockout. CONCLUSION: This study identifies PPAP2C as a key oncogenic driver and a robust independent prognostic biomarker in breast cancer. The findings provide compelling evidence that PPAP2C represents a promising therapeutic target, offering a new strategic avenue for precision therapy, particularly for aggressive breast cancer subtypes.

PLPP2↗

PubMed2026

GmMYB29 activates Gm4CL3 to enhance soybean resistance to Heterodera glycines.

Soybean cyst nematode is a devastating soil-borne pathogen that severely limits soybean yield worldwide. To uncover downstream target genes of the resistance-associated transcription factor GmMYB29, we combined ChIP-seq and RNA-seq data from T3-generation GmMYB29-overexpressing soybean plants, alongside Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses, to screen candidate genes carrying transcription factor binding peaks within the 2000 bp region upstream of transcription start sites (TSS). Four orthogonal molecular assays-yeast one-hybrid (Y1H), electrophoretic mobility shift assay (EMSA), dual-luciferase reporter (LUC) system, and GUS histochemical staining-collectively confirmed the specific physical interaction between GmMYB29 and the promoter of Gm4CL3. We generated transgenic soybean hairy roots overexpressing Gm4CL3 (OX-Gm4CL3) and CRISPR-Cas9-mediated Gm4CL3 knockout lines (KO-Gm4CL3), with wild-type (WT) plants serving as controls. Inoculation assays using SCN 3 demonstrated that OX-Gm4CL3 roots displayed substantially improved SCN resistance, while KO-Gm4CL3 roots were hypersusceptible to nematode infection. Mechanistic investigations revealed that Gm4CL3 promotes lignin deposition in root tissues to block SCN penetration. Furthermore, GmMYB29 and Gm4CL3 act synergistically to activate lignin biosynthetic pathways and strengthen soybean resistance against SCN 3 (SCN Race 3, the dominant physiological race in Northeast China). In summary, this study functionally characterizes Gm4CL3 and defines a previously unreported GmMYB29-Gm4CL3 regulatory cascade that mediates plant defense against SCN. This module functions independent of classic SCN resistance loci rhg1/Rhg4, providing new genetic resources for SCN-resistant soybean molecular breeding.

Glycine max↗

PubMed2025

Genetic Engineering and Screening Using Base Editing and Inducible Gene Knockout.

Genetic engineering and screening in human cells are powerful techniques for the precise and comprehensive identification and analysis of gene and protein domain functions. Genome-wide knockout screens have been extensively utilized to discover essential genes, tumor suppressors, and genes that regulate responses to various chemicals, including antimitotic and therapeutic drugs. The advent of base editors, which facilitate the targeted mutation of single amino acids, has advanced the identification of critical and functional domains or motifs. In this context, we outline methods for creating efficient base editor and inducible knockout cell lines for targeted gene manipulation and conducting genetic screens to elucidate the roles of genes and their domains within a specific cell biological context.

Humans↗

PubMed2025

Large-scale CRISPR screening in primary human 3D gastric organoids enables comprehensive dissection of gene-drug interactions.

Understanding how genes influence drug responses is critical for advancing personalized cancer treatments. However, identifying these gene-drug interactions in a physiologically relevant human system remains a challenge, as it requires a model that reflects the complexity and heterogeneity among individuals. Here we show that large-scale CRISPR-based genetic screens, including knockout, interference (CRISPRi), activation (CRISPRa), and single-cell approaches, can be applied in primary human 3D gastric organoids to systematically identify genes that affect sensitivity to cisplatin. Our screens uncover genes that modulate cisplatin response. By combining CRISPR perturbations with single-cell transcriptomics, we resolve how genetic alterations interact with cisplatin at the level of individual cells and uncover an unexpected link between fucosylation and cisplatin sensitivity. We identify TAF6L as a regulator of cell recovery from cisplatin-induced cytotoxicity. These results highlight the utility of human organoid models for dissecting gene-drug interactions and offer insights into therapeutic vulnerabilities in gastric cancer.

Humans↗