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: eLife

Article Title: C/EBPδ-induced epigenetic changes control the dynamic gene transcription of S100a8 and S100a9

doi: 10.7554/eLife.75594

Figure Lengend Snippet: ( A ) For genome-wide screen, over 100,000 plasmids, each containing a guide RNA towards different early consecutive exons, were packaged into lentiviral particles. Cas9-expressing ER-Hoxb8 cells were pool-transduced, selected, and differentiated to induce S100A9 expression. Hits and reference cells were collected by sorting according to their phenotypes of interest. DNA of both samples was purified for next-generation sequencing and subsequent analysis. ( B ) Precursor and differentiated Cas9 and Cas9-library ER-Hoxb8 cells were stained intracellularly for S100A9 using a FITC-labelled antibody. Cas9-library ER-Hoxb8 day 3 monocytes with no or lower S100A9 expression were sorted as hits, the remaining cells served as reference cells. ( C ) Data was analysed using the Model-based Analysis of Genome-wide CRISPR-Cas9 Knockout (MAGeCK) software for identification of enriched guide RNAs in the hit sample. Corresponding genes were rank-ordered by robust rank aggregation (RRA) scores. The list states the top 20 genes according to RRA scores, arranged after the number of guides that are enriched in the hit sample. See also and . Figure 1—source data 1. Gene summary of Model-based Analysis of Genome-wide CRISPR-Cas9 Knockout (MaGECK) analysis.

Article Snippet: Amplification of mouse CRISPR Knockout pooled library (GeCKO v2) in lentiGuide-Puro plasmid, purchased from AddGene (#1000000053) , was performed as described ( ).

Techniques: Genome Wide, Expressing, Purification, Next-Generation Sequencing, Staining, CRISPR, Knock-Out, Software

Journal: eLife

Article Title: C/EBPδ-induced epigenetic changes control the dynamic gene transcription of S100a8 and S100a9

doi: 10.7554/eLife.75594

Figure Lengend Snippet: Cell lines generated by CRISPR/Cas9 (A: PHF8, CSRP1, and HAND1 KO, dark grey bars = candidates from Genome-Scale CRISPR/Cas9 Knockout (GeCKO) screen, B: ATF3, STAT3, KLF5, IRF7, and C/EBPβ KO, light grey bars = candidates from previous studies) were compared to a non-targeting CRISPR/Cas9 control cell line. Cell lines generated from transgenic mice (C/EBPδ KO, black bars = hit from GeCKO screen) were compared to wildtype (WT) counterparts. All cells were differentiated to until day 2 or day 3 and relative S100a8 and S100a9 mRNA levels were analysed using quantitative reverse transcription polymerase chain reaction (qRT-PCR). Red lines indicate relative s100 level of controls (WT). Values are the means ± SEM of four experiments. *p<0.05, **p<0.01, ***p<0.001 by two-tailed Student’s t test.

Article Snippet: Amplification of mouse CRISPR Knockout pooled library (GeCKO v2) in lentiGuide-Puro plasmid, purchased from AddGene (#1000000053) , was performed as described ( ).

Techniques: Generated, CRISPR, Knock-Out, Control, Transgenic Assay, Reverse Transcription, Polymerase Chain Reaction, Quantitative RT-PCR, Two Tailed Test

Journal: eLife

Article Title: C/EBPδ-induced epigenetic changes control the dynamic gene transcription of S100a8 and S100a9

doi: 10.7554/eLife.75594

Figure Lengend Snippet:

Article Snippet: Amplification of mouse CRISPR Knockout pooled library (GeCKO v2) in lentiGuide-Puro plasmid, purchased from AddGene (#1000000053) , was performed as described ( ).

Techniques: Subcloning, Transfection, Construct, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Recombinant, Control, Genome Wide, CRISPR, Expressing, Plasmid Preparation, Marker, Sequencing, Cloning, Library Amplification, Mutagenesis, In Vitro, In Vivo, Purification, Protease Inhibitor, Immunoprecipitation, Protein Extraction, Isolation, Software