Genome-wide screening using CRISPR coupled with nuclease Cas9 (CRISPR/Cas9) is a powerful technology for the systematic evaluation of gene function. Statistically principled analysis is needed for the accurate identification of gene hits and associated pathways. Here, we describe how to perform computational analysis of CRISPR screens using the MAGeCKFlute pipeline. MAGeCKFlute combines the MAGeCK and MAGeCK-VISPR algorithms and incorporates additional downstream analysis functionalities. MAGeCKFlute is distinguished from other currently available tools by being a comprehensive pipeline that contains a series of functions for analyzing CRISPR screen data. This protocol explains how to use MAGeCKFlute to perform quality control, normalization, batch effect removal, copy number bias correction, gene hit identification, and downstream functional enrichment analysis for CRISPR screens. We also describe gene identification and data analysis in CRISPR screens involving drug treatment. Completing the entire MAGeCKFlute pipeline requires approximately two hours on a desktop computer running Linux or Mac OS and with R support. The MAGeCKFlute package is available at http://www.bioconductor.org/packages/release/bioc/html/MAGeCKFlute.html.
Purpose: Immune checkpoint blockade has shown remarkable efficacy, but in only a minority of patients with cancer, suggesting the need to develop additional treatment strategies. Aberrant glycosylation in tumors, resulting from the dysregulated expression of key enzymes in glycan biosynthesis, modulates the immune response. However, the role of glycan biosynthesis enzymes in antitumor immunity is poorly understood. We aimed to study the immunomodulatory effects of these enzymes.
A xylanase (Xyn10A) gene from the saline-alkali-tolerant microorganism Bacillus cellulosilyticus DSM 2522 was cloned and expressed in Escherichia coli BL21 (DE3). The open reading frame was composed of 1008 base pairs, and it encoded 335 amino acid residues belonging to glycosyl hydrolase family 10. The optimal temperature and pH of the purified Xyn10A were 40 °C and 8.0, respectively. The Xyn10A was sensitive to heat and showed obvious cold-adapted activity, retaining 38.3%, 55.7%, and 82.9% of the optimal activity at 4, 20, and 30 °C, respectively. Xyn10A also showed a high level of NaCl tolerance. The highest activity was observed with 1.5 M NaCl. The specific enzyme activity of Xyn10A was as much as 163.8 U/mg. Kinetic assays showed that Km, Vmax, and Kcat were 2.56 mg/mL, 202.5 μM/min/mg, and 132.6 /s, respectively. Additionally, the main hydrolysis products using birchwood xylan as substrate were xylobiose, xylotriose, and xylotetraose, as determined by thin layer chromatography analysis. As a cold-adapted and salt-tolerant enzyme, Xyn10A is an ideal candidate for further research and biotechnological applications.
BRAF is a serine/threonine kinase that harbors activating mutations in ∼7% of human malignancies and ∼60% of melanomas. Despite initial clinical responses to BRAF inhibitors, patients frequently develop drug resistance . To identify candidate therapeutic targets for BRAF inhibitor resistant melanoma , we conduct CRISPR screens in melanoma cells harboring an activating BRAF mutation that had also acquired resistance to BRAF inhibitors. To investigate the mechanisms and pathways enabling resistance to BRAF inhibitors in melanomas, we integrate expression, ATAC-seq, and CRISPR screen data. We identify the JUN family transcription factors and the ETS family transcription factor ETV5 as key regulators of CDK6 , which together enable resistance to BRAF inhibitors in melanoma cells. Our findings reveal genes contributing to resistance to a selective BRAF inhibitor PLX4720, providing new insights into gene regulation in BRAF inhibitor resistant melanoma cells.
The pleiotropic cytokine interferon-gamma (IFNγ) is associated with cytostatic, anti-proliferation, and pro-apoptotic functions in cancer cells. However, resistance to IFNγ occurs in many cancer cells, and the underlying mechanism is not fully understood. To investigate potential IFNγ resistance mechanisms, we performed IFNγ sensitivity screens in more than 40 cancer cell lines and characterized the sensitive and resistant cell lines. By applying CRISPR screening and transcriptomic profiling in both IFNγ-sensitive and IFNγ-resistant cells, we discovered that activation of double-strand break (DSB) repair genes could result in IFNγ resistance in cancer cells. Suppression of single-strand break (SSB) repair genes increased the dependency on DSB repair genes after IFNγ treatment. Furthermore, inhibition of the DSB repair pathway exhibited a synergistic effect with IFNγ treatment both in vitro and in vivo. The relationship between the activation of DSB repair genes and IFNγ resistance was further confirmed in clinical tumor profiles from The Cancer Genome Atlas (TCGA) and immune checkpoint blockade (ICB) cohorts. Our study provides comprehensive resources and evidence to elucidate a mechanism of IFNγ resistance in cancer and has the potential to inform combination therapies to overcome immunotherapy resistance.
<p>Sup.Table.S3: Gene sets enriched in Man2a1-null B16F10 tumors vs control tumors treated with anti-PD-L1. Pre-ranked gene set enrichment analysis (GSEA) was used to perform the analysis.</p>
<p>Sup.Fig.8: The effect of Man2a1 knockout on the immune cell infiltration.</p>
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.