Journal: Theranostics

Article Title: Truncated HDAC9 identified by integrated genome-wide screen as the key modulator for paclitaxel resistance in triple-negative breast cancer

doi: 10.7150/thno.44997

Figure Lengend Snippet: Integrated analyses of genome-Wide CRISPR/Cas9 screen and transcriptome sequencing. (A) Workflow of genome wide CRISPR/Cas9 knockout screening with PTX treatment. (B) Single guide RNA (sgRNA) read variations on day 14 after PTX treatment, compared to DMSO treatment. (C) Genes known to sensitize cellular response to PTX treatment. (D) Well-known resistant genes in response to PTX treatment. (E) Diagram illustrates construction of paclitaxel-resistant MDA-MB-231 cells. The cells were treated with 1 µM paclitaxel for 24 h, then changed to normal culture medium for 2 weeks. This procedure was repeated 12 times. (F) Bubble chart exhibiting significant differentially expressed genes with a log 2 -fold change (FC) ≤ -1 or ≥ 1. Bubble size represents the value of log 2 TPM in 231-PTX cells. (G) Triangle chart confirmed previously-reported genes playing a critical role in paclitaxel resistance in cancer. The size of the triangle represents the value of log 2 TPM in 231-PTX cells. (H) Distribution of the top 20 GSEA drug resistant-associated pathways: Taxol agent-related pathways constitute 25% of the total pathways. (I) One of the GSEA enrichment analyses among the top 20 Taxol-related pathways.

Article Snippet: The genome-wide CRISPR knockout (GeCKO v2) library was purchased from Addgene and was expanded to 1000× using an electronic transfection method.

Techniques: Genome Wide, CRISPR, Sequencing, Knock-Out

Journal: Theranostics

Article Title: Truncated HDAC9 identified by integrated genome-wide screen as the key modulator for paclitaxel resistance in triple-negative breast cancer

doi: 10.7150/thno.44997

Figure Lengend Snippet: Paclitaxel-sensitive/resistant candidates and their clinical prognostic values in breast cancer. (A-B) Volcano plot displays gene distribution of paclitaxel-sensitive (A) paclitaxel-resistant (B) candidates. X-axis represents log 2 -fold change of 231-PTX versus 231-WT and Y-axis represents log 10 p value of CRISPR/Cas9-positive (A) / (B) -negative screening. (C-F) Kaplan-Meier analysis of paclitaxel-sensitive candidates. Relapse-free survival Kaplan-Meier plots were based on gene expression, and the auto-select best cut-off was used to sort patients ( p < 0.05). (G-J) Kaplan-Meier plot of paclitaxel-resistant candidates. Relapse-free survival Kaplan-Meier plots were based on gene expression, and the autos-elect best cut-off was used to sort patients ( p < 0.05). (K-L) Cell growth after individual gene knock-out with single guide (sg) RNA was evaluated following treatment with 1 nM paclitaxel or DMSO for 6 d (*: p < 0.05; **: p < 0.01).

Article Snippet: The genome-wide CRISPR knockout (GeCKO v2) library was purchased from Addgene and was expanded to 1000× using an electronic transfection method.

Techniques: CRISPR, Gene Expression, Knock-Out

Journal: Cell reports

Article Title: Suppression of Ribosomal Pausing by eIF5A Is Necessary to Maintain the Fidelity of Start Codon Selection

doi: 10.1016/j.celrep.2019.10.129

Figure Lengend Snippet:

Article Snippet: Human CRISPR Knockout Pooled Library (GeCKO v2) , Addgene (Feng Zhang) , Pooled Library #1000000048, #1000000049 ( ) .

Techniques: Recombinant, Transfection, Infection, Cloning, Library Quantification, SYBR Green Assay, Mutagenesis, Reporter Assay, CRISPR, Plasmid Preparation, Knock-Out, Software

Journal: Cell reports

Article Title: Functional genomics identifies N-acetyllactosamine extension of complex N-glycans as a mechanism to evade lysis by natural killer cells.

doi: 10.1016/j.celrep.2024.114105

Figure Lengend Snippet: Figure 2. Validation of CRISPR screen hits (A) Experimental outline of the killing assay used to validate hits from the CRISPR screen. 221 cells expressing non-targeting sgRNA (sgNT) and sgRNA targeting genes of interest were pre-labeled with dyes and mixed at a 1:1 ratio before co-culture with NK cells. Surviving 221 cells were analyzed by flow cytometry. (B) Killing assays using activated NK cells and 221 target cells expressing sgRNAs for the indicated genes (green circles) compared to cells expressing sgNT (white circles). SPPL3 was deleted separately with two different sgRNAs. Each line represents an independent donor (n R 8, paired t test; n.s., not significant; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001). (C) NK cell degranulation induced by 221 cells expressing sgNT or sgRNAs targeting genes encoding the NK cell ligands MICB and CD58. Data shown as mean ± SEM (n = 6; *p < 0.05; ns, not significant).

Article Snippet: The GeCKO V2 human CRISPR knockout library from Addgene was then introduced into Endura Electrocompetent Cells by means of electroporation using a Bio-Rad Gene Pulser.

Techniques: Biomarker Discovery, CRISPR, Expressing, Labeling, Co-Culture Assay, Cytometry

Journal: Cell reports

Article Title: Functional genomics identifies N-acetyllactosamine extension of complex N-glycans as a mechanism to evade lysis by natural killer cells.

doi: 10.1016/j.celrep.2024.114105

Figure Lengend Snippet: Figure 6. Secondary CRISPR screen in SPPL3-deficient 221 cells to identify glycosyl transferases responsible for the inhibition of NK- mediated killing (A) CRISPR knockout screen in SPPL3-deleted 221 cells using a glycosylation-focused sgRNA sublibrary. 221 cells expressing the library were co-cultured with activated primary NK cells. Distribution of sgRNAs in surviving 221 cells was analyzed by NGS. Two biological repeats were plotted on the x axis and y axis, respectively. CRISPR beta scores were calculated using the MAGeCK MLE method. Enriched genes, signifying greater resistance, are shown in red, whereas depleted genes, signifying greater sensitivity, are in blue. Gene names of top hits are shown. (B) Ranked plot of cumulative CRISPR scores calculated by the score from SPPL3-knockout cells minus the score from 221 cells expressing sgNT. (C) Representative histograms of LEL binding to sgNT (top) or sgSPPL3 (bottom) 221 cells. (D) Representative histograms of LEL binding to SPPL3-knockout 221 cells expressing two individual sgRNAs with the first sgRNA being sgNT (top) or sgSPPL3 (bottom) and the second sgRNA either non-targeting or against B3GNT2. (E) NK cell degranulation stimulated by 221 cells expressing two individual sgRNAs as in (D). Each dot represents NK cells from an independent donor (n = 4, paired one-way ANOVA; ns not significant; *p < 0.05, **p < 0.01).

Article Snippet: The GeCKO V2 human CRISPR knockout library from Addgene was then introduced into Endura Electrocompetent Cells by means of electroporation using a Bio-Rad Gene Pulser.

Techniques: CRISPR, Inhibition, Knock-Out, Glycoproteomics, Expressing, Cell Culture, Binding Assay