human genome scale crispr knockout gecko v2 pooled library  (Addgene inc)

Journal: Environment international

Article Title: Genome-wide CRISPR-based screen identifies E2F transcription factor 1 as a regulator and therapeutic target of aristolochic acid-induced nephrotoxicity.

doi: 10.1016/j.envint.2024.109234

Figure Lengend Snippet: Fig. 1. Gene profiles of genome-wide CRISPR screen. A. Cells were treated with different doses of AAI for 48 h. After exposure, cell viability was measured by the MTS assay. B. Schematic diagram for genome-wide CRISPR screen. C. Volcano plot for candidate genes. D. Core analysis of candidate genes. E. Tox List for candidate genes. F. Disease and function plot for candidate genes. Data are presented as the mean ± SEM.

Article Snippet: The GeCKO v2 genome-scale CRISPR knockout library was obtained from Addgene (Plasmid #52961) and contains 65,383 single-guide RNAs (sgRNAs) targeting 19,050 genes.

Techniques: Genome Wide, CRISPR, MTS Assay

Journal: Molecular Cancer

Article Title: Genome-wide in vivo CRISPR screen identifies TGFβ3 as actionable biomarker of palbociclib resistance in triple negative breast cancer

doi: 10.1186/s12943-024-02029-4

Figure Lengend Snippet: In vivo genome-wide CRISPR knockout screen in TNBC. a Schematic representation of the approach used for gene discovery and validation. b Average tumor volume in NSG mice measured over 30 days. Intraperitoneal (i.p.) injections of either vehicle or palbociclib started on day 7 post-cell implantation, and lasted 23 days. Mean of three independent infection replicate experiments ( n = 6, 2 mice per biological replicate). Data are represented as mean ± standard deviation (SD). Significance was calculated using two-sided, unpaired t-test, p -value * < 0.05, ** < 0.01, *** < 0.001. c Principal component analysis (PCA) of the sgRNAs from the library sequenced in vehicle-treated tumors ( n = 6), and palbociclib-treated tumor samples ( n = 6) at day 30 after normalization. d 205 sgRNAs were enriched with log2-fold change (LFC) > 0 at false discovery rate (FDR) < 0.05 in palbociclib-treated tumors during the screen. Genes representing significant hits are highlighted in red. e Palbociclib sensitivity data was used to rank 38 breast cancer cell lines of varying subtypes, generating two profiles of cell lines, ‘sensitive’ and ‘resistant’. GSEA was used to determine whether 205 sgRNA gene set was significantly enriched in either group of cell lines. Enrichment plot provides the distribution of the enrichment score (green line) of the 205-gene set in the ranked cell lines (sensitive to resistant, left to right). The final, positive normalized enrichment score (NES) at 1.288 indicates significant enrichment of the 205-gene set at FDR < 0.25 in palbociclib ‘sensitive’ cell lines (FDR = 0.0568, p -value = 0.0568). f Using GSEA, expression levels of the 47 genes (core enrichment subset) are presented here. Cell lines are annotated with clinical information

Article Snippet: Human genome-scale CRISPR knockout pooled library (GeCKOv2, Addgene plasmid #1,000,000,048) was amplified according to manufacturer’s instructions and as shown previously [ ].

Techniques: In Vivo, Genome Wide, CRISPR, Knock-Out, Biomarker Discovery, Infection, Standard Deviation, Expressing

Journal: Molecular Cancer

Article Title: Genome-wide in vivo CRISPR screen identifies TGFβ3 as actionable biomarker of palbociclib resistance in triple negative breast cancer

doi: 10.1186/s12943-024-02029-4

Figure Lengend Snippet: In vivo validation of top candidate genes. a Gene modification detection of individual CRISPR-mediated knockouts of top candidate genes. b Cells transduced with non-targeting (NT) control or top candidate gene ( SLC40A1, TGFB3, SNRPN, ITGB6, BAMBI, TMEM176A or PDGFB, TMEM150A ) KO constructs were transplanted orthotopically into the mammary fat pads of NSG mice. Tumors were palpable before mice from each NT ( n = 10–22) or targeting group ( n = 10–12) were randomized into treatment groups (vehicle, n = 5–11; palbociclib (30 mg/kg), n = 5–11). Mean ± SD tumor volume is shown. Significance was calculated using two-sided, unpaired t-test, p -value ns. = nonsignificant, * < 0.05. c Tumor volumes of individual mice in each group, NT or targeting a candidate gene, either treated with vehicle or palbociclib at experiment endpoint ( n = 5). Midlines indicate median tumor volume. Significance was calculated using two-sided, unpaired t-test, p -value * < 0.05

Article Snippet: Human genome-scale CRISPR knockout pooled library (GeCKOv2, Addgene plasmid #1,000,000,048) was amplified according to manufacturer’s instructions and as shown previously [ ].

Techniques: In Vivo, Biomarker Discovery, Modification, CRISPR, Transduction, Control, Construct

Journal: Molecular Cancer

Article Title: Genome-wide in vivo CRISPR screen identifies TGFβ3 as actionable biomarker of palbociclib resistance in triple negative breast cancer

doi: 10.1186/s12943-024-02029-4

Figure Lengend Snippet: TGFβ3 potentiates palbociclib anti-tumor effect in vivo. a mRNA expression levels of TGFB1, TGFB2 and TGFB3 in SUM159PT following TGFB3 -specific overexpression using CRISPR activation (CRISPR/dCas9 SAM) ( n = 3). Data are represented as mean ± standard deviation (SD). Significance was calculated using two-sided, unpaired t-test, p -value * < 0.05. b Mice from control (lentiSAMv2) or TGFB3 -overexpressing (TGFB3g2 SAM) groups ( n = 13) were each randomized into treatment groups (vehicle, n = 6; palbociclib, n = 7). I.p. injections of the vehicle treatment or a low dose of palbociclib (10 mg/kg) were administered until study endpoint. Data are represented as mean ± SD. c Reduction in tumor growth presented for each group treated with palbociclib, lentiSAMv2 or TGFB3g2 SAM, as compared to the same groups treated with the vehicle. Data are represented as mean, at each timepoint. d left Tumor volumes of individual mice in each group at study endpoint. right Tumor weights of individual mice in each group at study endpoint. Midlines at median. Significance was calculated using ordinary, one-way ANOVA with Tukey’s multiple comparisons test, p -value * < 0.05, ** < 0.01, *** < 0.001. e Average mRNA expression levels of TGFB3 in tumors derived from the vehicle-treated control mice ( n = 6) and the TGFB3 -overexpressing mice ( n = 6). Data are represented as mean ± SD. Significance was calculated using two-sided, unpaired t-test, p -value * < 0.05, ** < 0.01, *** < 0.001. f Protein levels of TGFB3 (60 kDa) in tumors derived from the vehicle-treated control mice ( n = 6) and the TGFB3 -overexpressing mice ( n = 6). g Spontaneous metastasis to the lungs was assessed. Lung nodules were counted and compared in lungs derived from the vehicle-treated control mice ( n = 7) and the TGFB3 -overexpressing mice ( n = 6). Data represent metastatic nodule count per pair of lungs per mouse. Midlines at median. Significance was calculated using nonparametric Mann–Whitney U-test, p -value * < 0.05, ** < 0.01, *** < 0.001. h The effect of TGFB3 CRISPR-mediated knockout on lung colonization was assessed. Data represent metastatic nodule count per pair of lungs per mouse. Midlines at median. i Schematic representation of the use of recTGFβ3 in combination with palbociclib. MDA-MB-231 TNBC cells were transplanted into the mammary fat pads of NSG mice. Tumors were palpable before mice were randomized into treatment groups: vehicle, n = 9; recTGFβ3, n = 8; palbociclib, n = 8, combo (recTGFβ3 + palbociclib), n = 9. j Average tumor volume was measured over time. Data are represented as mean ± SD. k Tumor volumes of individual mice in each group at study endpoint. Midlines at median. Significance was calculated using ordinary, one-way ANOVA with Tukey’s multiple comparisons test, p -value * < 0.05. l Quantification of Ki67-positive cells stained by immunohistochemistry in tumor tissues from all four groups. Data are represented as mean ± SD ( n = 3–4). Significance was calculated using two-sided, unpaired t-test, p -value * < 0.05. m Representative images of Ki67 staining in two tumors per group

Article Snippet: Human genome-scale CRISPR knockout pooled library (GeCKOv2, Addgene plasmid #1,000,000,048) was amplified according to manufacturer’s instructions and as shown previously [ ].

Techniques: In Vivo, Expressing, Over Expression, CRISPR, Activation Assay, Standard Deviation, Control, Derivative Assay, MANN-WHITNEY, Knock-Out, Staining, Immunohistochemistry

Journal: International Journal of Biological Sciences

Article Title: Cullin3 deficiency shapes tumor microenvironment and promotes cholangiocarcinoma in liver-specific Smad4/Pten mutant mice

doi: 10.7150/ijbs.67379

Figure Lengend Snippet: Lentiviral delivery of the CRISPR knockout library identifies Cul3 as a suppressor of CC in SPC mice. (A) Outline of tail vein injection of lentivirus-mediated CRISPR knockout library. (B) Livers injected with the CRISPR knockout library develop tumors at 3-4 months, while the control livers are normal. (C) Summary of tumor numbers in each mouse. (D) Histology analysis of tumors induced by the CRISPR knockout library. AE1 indicates cholangiocytes and Hep Par1 indicates hepatocytes. PCNA represents cell proliferation. (E) Plot shows the average of sgRNA enrichment measured by high-throughput sequencing. (F) Outline of hydrodynamic tail vein injection of sgRNA. (G) Up panel: Representative images of the livers injected with sgRNAs targeting Smad4, Trp53 and Cul3. Down panel: Immunostaining of paraffin sections show the phenotype of CC. (H) Statistics of the tumor number, liver weight and the ratio of the liver weight to body weight (LW/BW).

Article Snippet: The following vectors were obtained from Addgene: pCDH-EF1-Luc2-P2A-copGFP (GFP-Luc, #72485), PX330 (#42230), lenti-CRISPR v2 (#52961) and the mouse genome-scale CRISPR knockout library (#1000000052).

Techniques: CRISPR, Knock-Out, Injection, Next-Generation Sequencing, Immunostaining