Exploring the molecular landscape of NNK-induced transformation: A comprehensive genome-wide CRISPR/Cas9 screening
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PROVIDER: S-EPMC10915513 | biostudies-literature | 2023 Sep
REPOSITORIES: biostudies-literature
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Project description:IntroductionArsenic trioxide (ATO) is a promising anticancer drug for hematological malignancy. Given the dramatic efficacy of acute promyelocytic leukemia (APL), ATO has been utilized in other types of cancers, including solid tumors. Unfortunately, the results were not comparable with the effects on APL, and the resistance mechanism has not been clarified yet. This study intends to identify relevant genes and pathways affecting ATO drug sensitivity through genome-wide CRISPR-Cas9 knockdown screening to provide a panoramic view for further study of ATO targets and improved clinical outcomes.MethodsA genome-wide CRISPR-Cas9 knockdown screening system was constructed for ATO screening. The screening results were processed with MAGeCK, and the results were subjected to pathway enrichment analysis using WebGestalt and KOBAS. We also performed protein-protein interaction (PPI) network analysis using String and Cytoscape, followed by expression profiling and survival curve analysis of critical genes. Virtual screening was used to recognize drugs that may interact with the hub gene.ResultsWe applied enrichment analysis and identified vital ATO-related pathways such as metabolism, chemokines and cytokines production and signaling, and immune system responses. In addition, we identified KEAP1 as the top gene relating to ATO resistance. We found that KEAP1 expression was higher in the pan-cancer, including ALL, than in normal tissue. Patients with acute myeloid leukemia (AML) with higher KEAP1 expression had worse overall survival (OS). A virtual screen showed that etoposide and eltrombopag could bind to KEAP1 and potentially interact with ATO.DiscussionATO is a multi-target anticancer drug, and the key pathways regulating its sensitivity include oxidative stress, metabolism, chemokines and cytokines, and the immune system. KEAP1 is the most critical gene regulating ATO drug sensitivity, which is related to AML prognosis and may bind to some clinical drugs leading to an interaction with ATO. These integrated results provided new insights into the pharmacological mechanism of ATO and potentiate for further applications in cancer treatments.
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Project description:Genome-wide screening is powerful method used to identify genes and pathways associated with a phenotype of interest. The simple eukaryote Dictyostelium discoideum has a unique life cycle and is often used as a crucial research model for a wide range of biological processes and rare metabolites. To address the inadequacies of conventional genetic screening approaches, we developed a highly efficient CRISPR/Cas9-based genome-wide screening system for Dictyostelium. A genome-wide library of 27,405 gRNAs and a kinase library of 4,582 gRNAs were compiled and mutant pools were generated. The resulting mutants were screened for defects in cell growth and more than 10 candidate genes were identified. Six of these were validated and five recreated mutants presented with growth abnormalities. Finally, the genes implicated in developmental defects were screened to identify the unknown genes associated with a phenotype of interest. These findings demonstrate the potential of the CRISPR/Cas9 system as an efficient genome-wide screening method.
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Project description:Loss-of-function screening by CRISPR/Cas9 gene knockout with pooled, lentiviral guide libraries is a widely applicable method for systematic identification of genes contributing to diverse cellular phenotypes. Here, Random Sequence Labels (RSLs) are incorporated into the guide library, which act as unique molecular identifiers (UMIs) to allow massively parallel lineage tracing and lineage dropout screening. RSLs greatly improve the reproducibility of results by increasing both the precision and the accuracy of screens. They reduce the number of cells needed to reach a set statistical power, or allow a more robust screen using the same number of cells.
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Project description:Apicomplexan parasites, such as Toxoplasma gondii, cause extensive morbidity and mortality in humans and livestock, highlighting the need for a deeper understanding of their molecular biology. Although techniques for the generation of targeted gene disruptions have long been available for apicomplexans, such methods are not readily scalable to the entire genome. We recently used CRISPR-Cas9 to disrupt all nuclear protein-coding genes in T. gondii using a pooled format. The method relies on transfection of a guide RNA library into parasites constitutively expressing Cas9. Here, we present the complete workflow of such a screen, including preparation of the guide RNA library, growth and testing of the recipient strain, generation of the mutant population, culture conditions for the screen, preparation of genomic DNA libraries, next-generation sequencing of the guide RNA loci, and analysis to detect fitness-conferring genes. This method can be deployed to study how culture conditions affect the repertoire of genes needed by parasites, which will enable studies of their metabolic needs, host specificity, and drug-resistance mechanisms. In addition, by manipulating the background in which the screen is performed, researchers will be able to investigate genetic interactions, which may help uncover redundancy or epistasis in the parasite genome. Using this method, a genome-wide screen and its analysis can be completed in 3 weeks, after ∼1 month of preparation to generate the library and grow the cells needed, making it a powerful tool for uncovering functionally important genes in apicomplexan parasites.
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Project description:BackgroundThe idiopathic pulmonary fibrosis (IPF) is a progressive and lethal interstitial lung disease with high morbidity and mortality. IPF is characterized by excessive extracellular matrix accumulation (ECM) and epithelial-mesenchymal transformation (EMT). To date, few anti-fibrotic therapeutics are available to reverse the progression of pulmonary fibrosis, and it is important to explore new profibrotic molecular regulators mediating EMT and pulmonary fibrosis.MethodsBased on our model of TGF-β1-induced EMT in BEAS-2B cells, we performed the genome-wide CRISPR/Cas9 knockout (GeCKO) screening technique, pathway and functional enrichment analysis, loss-of-function experiment, as well as other experimental techniques to comprehensively investigate profibrotic regulators contributing to EMT and the pathogenesis of pulmonary fibrosis.ResultsUtilizing the GeCKO library screening, we identified 76 top molecular regulators. Ten candidate genes were subsequently confirmed by integrating the high-throughput data with findings from pathway and functional enrichment analysis. Among the candidate genes, knockout of COL20A1 and COL27A1 led to decreased mRNA expression of ECM components (Fibronectin and Collagen-I), as well as an increased rate of cell apoptosis. The mRNA expression of Collagen-I, together with the cell viability and migration, were inhibited when knocking out the WNT11. In addition, a decrease in the protein deposition of ECM components was observed by suppressing the expression of COL20A1, COL27A1, and WNT11.ConclusionOur study demonstrates that the COL20A1, COL27A1, and WNT11 serve as key profibrotic regulators of EMT. Gaining understanding and insights into these key profibrotic regulators of EMT paves the way for the discovery of new therapeutic targets against the onset and progression of IPF.
| S-EPMC11872725 | biostudies-literature
Project description:Genome-wide clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated nuclease 9 (Cas9) screening is a simple screening method for locating loci under specific conditions, and it has been utilized in tumor drug resistance research for finding potential drug resistance-associated genes. This screening strategy has significant implications for further treatment of malignancies with acquired drug resistance. In recent years, studies involving genome-wide CRISPR/Cas9 screening have gradually increased. Here we review the recent application of genome-wide CRISPR/Cas9 screening for drug resistance, involving mitogen-activated protein kinase (MAPK) pathway inhibitors, poly (ADP-ribose) polymerase inhibitors (PARPi), alkylating agents, mitotic inhibitors, antimetabolites, immune checkpoint inhibitors (ICIs), and cyclin-dependent kinase inhibitors (CDKI). We summarize drug resistance pathways such as the KEAP1/Nrf2 pathway MAPK pathway, and NF-κB pathway. Also, we analyze the limitations and conditions for the application of genome-wide CRISPR/Cas9 screening techniques.
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Project description:Proteins evolve through the modular rearrangement of elements known as domains. Extant, multidomain proteins are hypothesized to be the result of domain accretion, but there has been limited experimental validation of this idea. Here, we introduce a technique for genetic minimization by iterative size-exclusion and recombination (MISER) for comprehensively making all possible deletions of a protein. Using MISER, we generate a deletion landscape for the CRISPR protein Cas9. We find that the catalytically-dead Streptococcus pyogenes Cas9 can tolerate large single deletions in the REC2, REC3, HNH, and RuvC domains, while still functioning in vitro and in vivo, and that these deletions can be stacked together to engineer minimal, DNA-binding effector proteins. In total, our results demonstrate that extant proteins retain significant modularity from the accretion process and, as genetic size is a major limitation for viral delivery systems, establish a general technique to improve genome editing and gene therapy-based therapeutics.
| S-EPMC8476515 | biostudies-literature
Project description:BackgroundGenome-wide CRISPR-Cas9 dropout screens can identify genes whose knockout affects cell viability. Recent CRISPR screens detected thousands of essential genes required for cellular survival and key cellular processes; however discovering novel lineage-specific genetic dependencies from the many hits still remains a challenge.ResultsTo assess whether CRISPR-Cas9 dropout screens can help identify cancer dependencies, we screened two human cancer cell lines carrying known and distinct oncogenic mutations using a genome-wide sgRNA library. We found that the gRNA targeting the driver mutation EGFR was one of the highest-ranking candidates in the EGFR-mutant HCC-827 lung adenocarcinoma cell line. Likewise, sgRNAs for NRAS and MAP2K1 (MEK1), a downstream kinase of mutant NRAS, were identified among the top hits in the NRAS-mutant neuroblastoma cell line CHP-212. Depletion of these genes targeted by the sgRNAs strongly correlated with the sensitivity to specific kinase inhibitors of the EGFR or RAS pathway in cell viability assays. In addition, we describe other dependencies such as TBK1 in HCC-827 cells and TRIB2 in CHP-212 cells which merit further investigation.ConclusionsWe show that genome-wide CRISPR dropout screens are suitable for the identification of oncogenic drivers and other essential genes.
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Project description:Glioblastomas (GBM) is the most common primary malignant brain tumor, and radiotherapy plays a critical role in its therapeutic management. Unfortunately, the development of radioresistance is universal. Here, we identified calcium-regulated heat-stable protein 1 (CARHSP1) as a critical driver for radioresistance utilizing genome-wide CRISPR activation screening. This is a protein with a cold-shock domain (CSD)-containing that is highly similar to cold-shock proteins. CARHSP1 mRNA level was upregulated in irradiation-resistant GBM cells and knockdown of CARHSP1 sensitized GBM cells to radiotherapy. The high expression of CARHSP1 upon radiation might mediate radioresistance by activating the inflammatory signaling pathway. More importantly, patients with high levels of CARHSP1 had poorer survival when treated with radiotherapy. Collectively, our findings suggested that targeting the CARHSP1/TNF-α inflammatory signaling activation induced by radiotherapy might directly affect radioresistance and present an attractive therapeutic target for GBM, particularly for patients with high levels of CARHSP1.
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Project description:The adenosine analogue remdesivir has emerged as a front-line antiviral treatment for SARS-CoV-2, with preliminary evidence that it reduces the duration and severity of illness1.Prior clinical studies have identified adverse events1,2, and remdesivir has been shown to inhibit mitochondrial RNA polymerase in biochemical experiments7, yet little is known about the specific genetic pathways involved in cellular remdesivir metabolism and cytotoxicity. Through genome-wide CRISPR-Cas9 screening and RNA sequencing, we show that remdesivir treatment leads to a repression of mitochondrial respiratory activity, and we identify five genes whose loss significantly reduces remdesivir cytotoxicity. In particular, we show that loss of the mitochondrial nucleoside transporter SLC29A3 mitigates remdesivir toxicity without a commensurate decrease in SARS-CoV-2 antiviral potency and that the mitochondrial adenylate kinase AK2 is a remdesivir kinase required for remdesivir efficacy and toxicity. This work elucidates the cellular mechanisms of remdesivir metabolism and provides a candidate gene target to reduce remdesivir cytotoxicity.
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