Search Results (75)

Conventional methods for generating knock-out or knock-in mammalian cell models using CRISPR-Cas9 genome editing often require tedious single-cell clone selection and expansion. In this study, we develop and optimise rapid and robust strategies to engineer homozygous fluorescent reporter knock-in cell pools with precise genome editing, circumventing clonal variability inherent to traditional approaches. To reduce false-positive cells associated with random integration, we optimise the design of donor DNA by removing the start codon of the fluorescent reporter and incorporating a self-cleaving T2A peptide system. Using fluorescence-assisted cell sorting (FACS), we efficiently identify and isolate the desired homozygous fluorescent knock-in clones, establishing stable cell pools that preserve parental cell line heterogeneity and faithfully reflect endogenous transcriptional regulation of the target gene. We evaluate the knock-in efficiency and rate of undesired random integration in the electroporation method with either a dual-plasmid system (sgRNA and donor DNA in two separate vectors) or a single-plasmid system (sgRNA and donor DNA combined in one vector). We further demonstrate that coupling our single-plasmid construct with an integrase-deficient lentivirus vector (IDLV) packaging system efficiently generates fluorescent knock-in reporter cell pools, offering flexibility between electroporation and lentivirus transduction methods. Notably, compared to the electroporation methods, the IDLV system significantly minimises random integration. Moreover, the resulting reporter cell lines are compatible with most of the available genome-wide sgRNA libraries, enabling unbiased CRISPR screens to identify key transcriptional regulators of a gene of interest. Overall, our methodologies provide a powerful genetic tool for rapid and robust generation of fluorescent reporter knock-in cell pools with precise genome editing by CRISPR-Cas9 for various research purposes. Full article

Viral oncolysis is considered a promising cancer treatment method because of its good tolerability and durable anti-tumor effects. Compared with other oncolytic viruses, Newcastle disease virus (NDV) has some distinct advantages. As an RNA virus, NDV does not recombine with the host genome, making it safer compared with DNA viruses and retroviruses; NDV can induce syncytium formation, allowing the virus to spread among cells without exposure to host neutralizing antibodies; and its genome adheres to the hexamer genetic code rule (genome length as a multiple of six nucleotides), ensuring accurate replication, low recombination rates, and high genetic stability. Although wild-type NDV has a killing effect on various tumor cells, its oncolytic effect and working mechanism are diverse, increasing the complexity of generating engineered oncolytic viruses with NDV. This study aims to employ whole-genome CRISPR-Cas9 knockout screening and RNA sequencing to identify putative key regulatory factors involved in the interaction between NDV and human colon cancer HCT116 cells and map their global interaction networks. The results suggests that NDV infection disrupts cellular homeostasis, thereby exerting oncolytic effects by inhibiting cell metabolism and proliferation. Meanwhile, the antiviral immune response triggered by NDV infection, along with the activation of anti-apoptotic signaling pathways, may be responsible for the limited oncolytic efficacy of NDV against HCT116 cells. These findings not only enhance our understanding of the oncolytic mechanism of NDV against colonic carcinoma but also provide potential strategies and targets for the development of NDV-based engineered oncolytic viruses. Full article

Background/Objectives: The treatment of multiple myeloma (MM) remains a challenge, as almost all patients will eventually relapse. Proteasome inhibitors are a cornerstone in the management of MM. Unfortunately, validated biomarkers predicting drug response are largely missing. Therefore, we aimed to identify genes associated with drug resistance or sensitization to proteasome inhibitors. Methods: We performed genome-wide CRISPR-Cas9 knockout (KO) screens in human KMS-28-BM myeloma cells to identify genetic determinants associated with resistance or sensitization to proteasome inhibitors. Results: We show that KO of KLF13 and PSMC4 induces drug resistance, while NUDCD2, OSER1 and HERC1 KO cause drug sensitization. Subsequently, we focused on top sensitization hit, NUDCD2, which acts as a co-chaperone of Hsp90 to regulate the LIS1/dynein complex. RNA sequencing showed downregulation of genes involved in the ERAD pathway and in ER-associated ubiquitin-dependent protein catabolic processes in both untreated and carfilzomib-treated NUDCD2 KO cells, suggesting that NUDCD2 depletion alters protein degradation. Furthermore, bortezomib-treated NUDCD2 KO cells showed a decreased expression of genes that have a function in oxidative phosphorylation and the mitochondrial membrane, such as Carnitine Palmitoyltransferase 1A (CPT1A). CPT1A catalyzes the uptake of long chain fatty acids into mitochondria. Mitochondrial lipid metabolism has recently been reported as a possible therapeutic target for MM drug sensitivity. Conclusions: These results contribute to the search for therapeutic targets that can sensitize MM patients to proteasome inhibitors. Full article

Seed development plays a critical role in determining both crop yield and grain quality in rice. As a key nutrient storage organ, the rice endosperm development not only contributes to grain filling but also plays an essential role during the early stages of seed germination. Amino acid metabolism is active during the process of seed development and seed germination. Asparagine is a primary amino acid responsible for long-distance organic nitrogen transport in plants. Asparagine synthetase catalyzes the synthesis of asparagine from aspartate and glutamine. In this study, CRISPR/Cas9-mediated knockout mutants of the OsASN2 gene of rice were generated. Homozygous mutants exhibited complete failure of seed germination, and heterozygotes could not produce homozygous offspring. Endosperm development of homozygous mutant seeds showed severe defects. Additionally, interacting protein screening combined with pull-down and co-immunoprecipitation (Co-IP) assays confirmed that OsASN2 physically interacted with pyruvate phosphate dikinase OsPPDKB, the mutants of which showed impaired endosperm development. These findings collectively indicate that OsASN2 plays a critical role in seed development and germination in rice. Full article

With the groundbreaking advancements in genome editing technologies, particularly CRISPR-Cas9, creating knockout mutants has become highly efficient. However, the CRISPR-Cas9 system introduces DNA double-strand breaks, increasing the risk of chromosomal rearrangements and posing a major obstacle to simultaneous multiple gene knockout. Base-editing systems, such as Target-AID, are safe alternatives for precise base modifications without requiring DNA double-strand breaks, serving as promising solutions for existing challenges. Nevertheless, the absence of adequate tools to support Target-AID-based gene knockout highlights the need for a comprehensive system to design guide RNAs (gRNAs) for the simultaneous knockout of multiple genes. Here, we aimed to develop KOnezumi-AID, a command-line tool for gRNA design for Target-AID-mediated genome editing. KOnezumi-AID facilitates gene knockout by inducing the premature termination codons or promoting exon skipping, thereby generating experiment-ready gRNA designs for mouse and human genomes. Additionally, KOnezumi-AID exhibits batch processing capacity, enabling rapid and precise gRNA design for large-scale genome editing, including CRISPR screening. In summary, KOnezumi-AID is an efficient and user-friendly tool for gRNA design, streamlining genome editing workflows and advancing gene knockout research. Full article

Background/Objectives: Early and aggressive metastasis is a major feature of pancreatic ductal adenocarcinoma. Understanding the processes underlying metastasis is crucial for making a difference to disease outcome. Towards these ends, we looked in a comprehensive manner for genes that are metastasis-specific. Methods: A genome-wide CRISPR-Cas9 gene knockout screen with 259,900 single guide RNA constructs was performed on pancreatic cancer cell lines with very high or very low metastatic capacity, respectively. Functional aspects of some of the identified genes were analysed in vitro. The injection of tumour cells with or without a gene knockout into mice was used to confirm the effect on metastasis. Results: The knockout of 590 genes—and, with higher analysis stringency, 67 genes—affected the viability of metastatic cells substantially, while these genes were not vital to non-metastasizing cells. Further evaluations identified different molecular processes related to this observation. One of the genes was MYBL2, encoding for a well-known transcription factor involved in the regulation of cell survival, proliferation, and differentiation in cancer tissues. In our metastasis-focussed study, no novel functional activity was detected for MYBL2, however. Instead, a metastasis-specific transformation of its genetic interaction with FOXM1 was observed. The interaction was synergistic in cells of low metastatic capacity, while there was a strong switch to a buffering mode in metastatic cells. In vivo analyses confirmed the strong effect of MYBL2 on metastasis. Conclusions: The genes found to be critical for the viability of metastatic cells form a basis for further investigations of the processes responsible for triggering and driving metastasis. As shown for MYBL2, unexpected processes of regulating metastasis might also be involved. Full article

Erythropoietic protoporphyria (EPP1) results in painful photosensitivity and severe liver damage in humans due to the accumulation of fluorescent protoporphyrin IX (PPIX). While zebrafish (Danio rerio) models for porphyria exist, the utility of ferrochelatase (fech) knockout zebrafish, which exhibit EPP, for therapeutic screening and biological studies remains unexplored. This study investigated the use of clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9-mediated fech-knockout zebrafish larvae as a model of EPP1 for drug screening. CRISPR/Cas9 was employed to generate fech-knockout zebrafish larvae exhibiting morphological defects without lethality prior to 9 days post-fertilization (dpf). To assess the suitability of this model for drug screening, ursodeoxycholic acid (UDCA), a common treatment for cholestatic liver disease, was employed. This treatment significantly reduced PPIX fluorescence and enhanced bile-secretion-related gene expression (abcb11a and abcc2), indicating the release of PPIX. Acridine orange staining and quantitative reverse transcription polymerase chain reaction analysis of the bax/bcl2 ratio revealed apoptosis in fech^−/− larvae, and this was reduced by UDCA treatment, indicating suppression of the intrinsic apoptosis pathway. Neutral red and Sudan black staining revealed increased macrophage and neutrophil production, potentially in response to PPIX-induced cell damage. UDCA treatment effectively reduced macrophage and neutrophil production, suggesting its potential to alleviate cell damage and liver injury in EPP1. In conclusion, CRISPR/Cas9-mediated fech^−/− zebrafish larvae represent a promising model for screening drugs against EPP1. Full article

The recurrence of diffuse large B-cell lymphoma (DLBCL) has been observed in 40% of cases. The standard of care for refractory/relapsed DLBCL (RR-DLBCL) is platinum-based treatment prior to autologous stem cell transplantation; however, the prognosis for RR-DLBCL patients remains poor. Thus, to identify genes affecting the cisplatin response in DLBCL, cisplatin-based whole-genome CRISPR-Cas9 knockout screens were performed in this study. We discovered DNA damage response (DDR) pathways as enriched among identified sensitizing CRISPR-mediated gene knockouts. In line, the knockout of the nucleotide excision repair genes XPA and ERCC6 sensitized DLBCL cells to platinum drugs irrespective of proliferation rate, thus documenting DDR as essential for cisplatin sensitivity in DLBCL. Functional analysis revealed that the loss of XPA and ERCC6 increased DNA damage levels and altered cell cycle distribution. Interestingly, we also identified BTK, which is involved in B-cell receptor signaling, to affect cisplatin response. The knockout of BTK increased cisplatin sensitivity in DLBCL cells, and combinatory drug screens revealed a synergistic effect of the BTK inhibitor, ibrutinib, with platinum drugs at low concentrations. Applying local and external DLBCL cohorts, we addressed the clinical relevance of the genes identified in the CRISPR screens. BTK was among the most frequently mutated genes with a frequency of 3–5%, and XPA and ERCC6 were also mutated, albeit at lower frequencies. Furthermore, 27–54% of diagnostic DLBCL samples had mutations in pathways that can sensitize cells to cisplatin. In conclusion, this study shows that XPA and ERCC6, in addition to BTK, are essential for the response to platinum-based drugs in DLBCL. Full article

The spatiotemporal expression patterns of genes are crucial for maintaining normal physiological functions in animals. Conditional gene knockout using the cyclization recombination enzyme (Cre)/locus of crossover of P1 (Cre/LoxP) strategy has been extensively employed for functional assays at specific tissue or developmental stages. This approach aids in uncovering the associations between phenotypes and gene regulation while minimizing interference among distinct tissues. Various Cre-engineered mouse models have been utilized in the male reproductive system, including Dppa3-MERCre for primordial germ cells, Ddx4-Cre and Stra8-Cre for spermatogonia, Prm1-Cre and Acrv1-iCre for haploid spermatids, Cyp17a1-iCre for the Leydig cell, Sox9-Cre for the Sertoli cell, and Lcn5/8/9-Cre for differentiated segments of the epididymis. Notably, the specificity and functioning stage of Cre recombinases vary, and the efficiency of recombination driven by Cre depends on endogenous promoters with different sequences as well as the constructed Cre vectors, even when controlled by an identical promoter. Cre mouse models generated via traditional recombination or CRISPR/Cas9 also exhibit distinct knockout properties. This review focuses on Cre-engineered mouse models applied to the male reproductive system, including Cre-targeting strategies, mouse model screening, and practical challenges encountered, particularly with novel mouse strains over the past decade. It aims to provide valuable references for studies conducted on the male reproductive system. Full article

Thousands of lncRNAs have been found in zebrafish embryogenesis and adult tissues, but their identification and organogenesis-related functions have not yet been elucidated. In this study, high-throughput sequencing was performed at three different organogenesis stages of zebrafish embryos that are important for zebrafish muscle development. The three stages were 10 hpf (hours post fertilization) (T1), 24 hpf (T2), and 36 hpf (T3). LncRNA gas5, associated with muscle development, was screened out as the next research target by high-throughput sequencing and qPCR validation. The spatiotemporal expression of lncRNA gas5 in zebrafish embryonic muscle development was studied through qPCR and in situ hybridization, and functional analysis was conducted using CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/Cas9, CRISPR/Cas9). The results were as follows: (1) A total of 1486 differentially expressed lncRNAs were identified between T2 and T1, among which 843 lncRNAs were upregulated and 643 were downregulated. The comparison with T3 and T2 resulted in 844 differentially expressed lncRNAs, among which 482 lncRNAs were upregulated and 362 lncRNAs were downregulated. A total of 2137 differentially expressed lncRNAs were found between T3 and T1, among which 1148 lncRNAs were upregulated and 989 lncRNAs were downregulated, including lncRNA gas5, which was selected as the target gene. (2) The results of spatiotemporal expression analysis showed that lncRNA gas5 was expressed in almost all detected embryos of different developmental stages (0, 2, 6, 10, 16, 24, 36, 48, 72, 96 hpf) and detected tissues of adult zebrafish. (3) After lncRNA gas5 knockout using CRISPR/Cas9 technology, the expression levels of detected genes related to muscle development and adjacent to lncRNA gas5 were more highly affected in the knockout group compared with the control group, suggesting that lncRNA gas5 may play a role in embryonic muscle development in zebrafish. (4) The results of the expression of the skeletal myogenesis marker myod showed that the expression of myod in myotomes was abnormal, suggesting that skeletal myogenesis was affected after lncRNA gas5 knockout. The results of this study provide an experimental basis for further studies on the role of lncRNA gas5 in the embryonic skeletal muscle development of zebrafish. Full article

Follicle-stimulating hormone (FSH) regulates ovarian follicle development through specific gene expression programs. Granulosa cells (GCs) are somatic cells surrounding the oocytes, secreting gonadotropins to regulate ovulation and promote follicular development. By analyzing the effects of different doses of FSH on the proliferation of GCs, we found that adding 10 ng/mL of FSH, as the optimal concentration, could promote the growth of GCs. Furthermore, we have successfully constructed the first CRISPR-Cas9 knockout library targeting the genes on chromosomes 2 and 3 and the X chromosomes of the sheep massively parallel coding gene, as well as an ovarian GCs knockout cell library. For the first time, we have exposed the knockout cell library to a concentration of 10 ng/mL FSH to explore the underlying mechanisms. Through this screening, we have identified 836 positive–negative screening genes that are responsive to FSH, thereby revealing the regulatory mechanisms and screening the functionality of candidate genes. Next, RNA-Seq of control (0 ng/mL), low (10 ng/mL), and high (100 ng/mL) doses of FSH revealed 1708 differentially expressed genes, and combined with 836 genes, we obtained 129 FSH dose-dependent genes with extremely significant differences. This enables us to delve deeper into investigating and identifying the mechanisms by which FSH regulates GCs. More generally, we have discovered new regulatory factors and identified reproductivity-associated major effectors. These findings provide novel research directions for further studies on sheep reproduction. Full article

Mosquitoes transmit a range of pathogens, causing devastating effects on human health. Population genetic control strategies have been developed and successfully used for several mosquito species. The most important step in identifying potential targets for mosquito control is the understanding of gene function. RNA interference (RNAi) is a powerful tool for gene silencing which has been widely used to study gene function in insects via knockdown of expression. The success of RNAi in insects depends on the efficient delivery of dsRNA into the cells, with microinjections being the most commonly used to study mosquito gene function. However, microinjections in the pupal stage lead to significant mortality in Aedes and Culex species, and few studies have performed microinjections in Culicinae pupae. Advanced techniques, such as CRISPR/Cas9 knockout, require establishing individual mosquito lines for each gene studied, and maintaining such lines may be limited by the insect-rearing capacity of a laboratory. Moreover, at times gene knockout during early development (embryo stage) has a deleterious effect on mosquito development, precluding the analysis of gene function in the pupal and adult stages and its potential for mosquito control. There is a need for a simple procedure that can be used for the fast and reliable examination of adult gene function via RNAi knockdown. Here, we focus on the aquatic stages of the mosquito life cycle and suggest a quick and easy assay for screening the functional role of genes in Culex pipiens mosquitoes without using microinjections. By dehydration of early stage pupae and subsequent rehydration in highly concentrated dsRNA, we achieved a moderate knockdown of laccase 2, a gene that turns on in the pupal stage and is responsible for melanization and sclerotization of the adult cuticle. Full article

The integration of viral DNA into a host genome is an important step in HIV-1 replication. However, due to the high failure rate of integration, the majority of viral DNA exists in an unintegrated state during HIV-1 infection. In contrast to the robust expression from integrated viral DNA, unintegrated HIV-1 DNA is very poorly transcribed in infected cells, but the molecular machinery responsible for the silencing of unintegrated HIV-1 DNA remains poorly characterized. In this study, we sought to characterize new host factors for the inhibition of expression from unintegrated HIV-1 DNA. A genome-wide CRISPR-Cas9 knockout screening revealed the essential role of phosphatase and tensin homolog (PTEN) in the silencing of unintegrated HIV-1 DNA. PTEN’s phosphatase activity negatively regulates the PI3K-Akt pathway to inhibit the transcription from unintegrated HIV-1 DNA. The knockout (KO) of PTEN or inhibition of PTEN’s phosphatase activity by point mutagenesis activates Akt by phosphorylation and enhances the transcription from unintegrated HIV-1 DNA. Inhibition of the PI3K-Akt pathway by Akt inhibitor in PTEN-KO cells restores the silencing of unintegrated HIV-1 DNA. Transcriptional factors (NF-κB, Sp1, and AP-1) are important for the activation of unintegrated HIV-1 DNA in PTEN-KO cells. Finally, the knockout of PTEN increases the levels of active epigenetic marks (H3ac and H3K4me3) and the recruitment of PolII on unintegrated HIV-1 DNA chromatin. Our experiments reveal that PTEN targets transcription factors (NF-κB, Sp1, and AP-1) by negatively regulating the PI3K-Akt pathway to promote the silencing of unintegrated HIV-1 DNA. Full article

Human antigen R (HuR) is an RNA-binding protein that regulates the post-transcriptional reaction of its target mRNAs. HuR is a critical factor in cancer development and has been identified as a potential target in many cancer models. It participates in the viral life cycle by binding to viral RNAs. In prior work, we used CRISPR/Cas9 screening to identify HuR as a prospective host factor facilitating Japanese encephalitis virus (JEV) infection. The HuR gene was successfully knocked out in U251 cell lines using the CRISPR/Cas9 gene-editing system, with no significant difference in cell growth between U251-WT and U251-HuR-KO2 cells. Here, we experimentally demonstrate for the first time that the knockout of the HuR gene inhibits the replication ability of JEV in U251 cell lines. These results play an essential role in regulating the replication level of JEV and providing new insights into virus–host interactions and potential antiviral strategies. It also offers a platform for investigating the function of HuR in the life cycle of flaviviruses. Full article

The protein phosphatase PP2C plays an important role in plant responses to stress. Therefore, the identification of maize PP2C genes that respond to drought stress is particularly important for the improvement and creation of new drought-resistant assortments of maize. In this study, we identified 102 ZmPP2C genes in maize at the genome-wide level. We analyzed the physicochemical properties of 102 ZmPP2Cs and constructed a phylogenetic tree with Arabidopsis. By analyzing the gene structure, conserved protein motifs, and synteny, the ZmPP2Cs were found to be strongly conserved during evolution. Sixteen core genes involved in drought stress and rewatering were screened using gene co-expression network mapping and expression profiling. The qRT-PCR results showed 16 genes were induced by abscisic acid (ABA), drought, and NaCl treatments. Notably, ZmPP2C15 exhibited a substantial expression difference. Through genetic transformation, we overexpressed ZmPP2C15 and generated the CRISPR/Cas9 knockout maize mutant zmpp2c15. Overexpressing ZmPP2C15 in Arabidopsis under drought stress enhanced growth and survival compared with WT plants. The leaves exhibited heightened superoxide dismutase (SOD), peroxidase (POD), ascorbate peroxidase (APX), and catalase (CAT) activities, elevated proline (Pro) content, and reduced malondialdehyde (MDA) content. Conversely, zmpp2c15 mutant plants displayed severe leaf dryness, curling, and wilting under drought stress. Their leaf activities of SOD, POD, APX, and CAT were lower than those in B104, while MDA was higher. This suggests that ZmPP2C15 positively regulates drought tolerance in maize by affecting the antioxidant enzyme activity and osmoregulatory substance content. Subcellular localization revealed that ZmPP2C15 was localized in the nucleus and cytoplasm. Yeast two-hybrid (Y2H) and bimolecular fluorescence complementation (BiFC) experiments demonstrated ZmPP2C15’s interaction with ZmWIN1, ZmADT2, ZmsodC, Zmcab, and ZmLHC2. These findings establish a foundation for understanding maize PP2C gene functions, offering genetic resources and insights for molecular design breeding for drought tolerance. Full article