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

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: Science Advances

Article Title: Genome-wide CRISPR-Cas9 screen identified KLF11 as a druggable suppressor for sarcoma cancer stem cells

doi: 10.1126/sciadv.abe3445

Figure Lengend Snippet: ( A ) Experimental design of an eOct4-EGFP reporter–based genome-wide CRISPR screen. ( B ) Volcano plot displaying the log 2 fold change and adjusted P value for all sgRNAs identified in the screen. Negative regulators with a threshold of FDR < 0.05 are labeled as small dark gray dots, selected genes with known function as large blue and orange dots, and KLF11 as large red dot. ( C ) qRT-PCR analysis of KLF11 expression in CD133 + and CD133 − cells from primary osteosarcoma cells ( n = 3). ( D ) qRT-PCR analysis of KLF11 expression in spheres and adherent cells from primary osteosarcoma cells ( n = 3). ( E ) Immunofluorescence detection of KLF11 (red) in primary osteosarcoma #4 spheres and adherent cells. The nuclei were stained with 4,6-diamidino-2-phenylindole (DAPI) (blue). Scale bar, 25 μm. ( F ) In vivo limiting dilution assay of KLF11-deleted and ectopic KLF11 rescue MG63 cells, and KLF11-knockdown primary osteosarcoma cells (sample #4). KO, knockout. n = 6 for each group. ( G ) In vivo limiting dilution assay of KLF11-overexpressing and control osteosarcoma spheres (143B and SJSA-1). n = 6 for each group. ( H ) Sphere formation assay of KLF11-knockdown primary osteosarcoma cell. ( I ) Sphere formation assay of KLF11-overexpressing primary osteosarcoma cells ( n = 3). ( J ) Incidences of tumorigenesis of CD133 + SJSA-1 cells infected with indicated adenovirus in serial transplantation models. ** P < 0.01 compared with untreated CD133 + SJSA-1 cells in the first inoculation by Fisher’s exact test. ( K ) Incidences of tumorigenesis of CD45 − CD133 + primary osteosarcoma cells (sample #10) infected with indicated adenovirus in serial transplantation models. ** P < 0.01 compared with untreated primary osteosarcoma cells in the first inoculation by Fisher’s exact test.

Article Snippet: The human genome-scale CRISPR knockout library (GeCKO v2, Addgene #1000000048) in the lentiCRISPR v2 vector (Addgene #52961) consists of 123,411 sgRNAs that target 19,050 protein-coding genes (6 sgRNAs per gene) and 1864 microRNAs (4 sgRNAs per microRNA) and also includes around 1000 nontargeting control sgRNAs ( , ).

Techniques: Genome Wide, CRISPR, Labeling, Quantitative RT-PCR, Expressing, Immunofluorescence, Staining, In Vivo, Limiting Dilution Assay, Knockdown, Knock-Out, Control, Tube Formation Assay, Infection, Transplantation Assay

Journal: The FASEB Journal

Article Title: Genome-wide CRISPR knockout screens identify NCAPG as an essential oncogene for hepatocellular carcinoma tumor growth

doi: 10.1096/fj.201802213RR

Figure Lengend Snippet: Clinically relevant targets identified from the CRISPR cell growth screen

Article Snippet: The human Genome-Scale CRISPR Knock-Out (GeCKO) v2 CRISPR knockout pooled library was a gift from Feng Zhang (Broad Institute, Massachusetts Institute of Technology, Cambridge, MA, USA) (Addgene 1000000048).

Techniques: CRISPR, Microarray

Journal: The FASEB Journal

Article Title: Genome-wide CRISPR knockout screens identify NCAPG as an essential oncogene for hepatocellular carcinoma tumor growth

doi: 10.1096/fj.201802213RR

Figure Lengend Snippet: The clinically relevant targets from the CRISPR screen are enriched in the cell cycle pathway. A ) Volcano plot showing significantly overexpressed genes (red) and underexpressed genes (green) between 70 HCC tumors and 37 nontumor samples. Significantly differentially expressed genes were indicated in the 2 upper lateral quadrants with absolute FC >2 and q < 0.05. B ) Top: Venn diagram showing 13 clinically relevant targets by overlapping 795 common hits from the depletion screen with 423 significantly overexpressed genes in HCC tumors. Bottom: ingenuity pathway analysis showing all 13 clinically relevant targets (in bold) that participated in the interplay of cell cycle and cell death and survival networks. C ) KEGG pathway enrichment analysis showing significant enrichment in cell cycle among the 13 clinically relevant targets. D ) Pearson correlation matrix of the 13 clinically relevant targets showing a tight cluster of MAD2L1, CCNA2, NCAPG, and BUB1B (black box) in the HCC microarray dataset shown in A . ANXA2, annexin A2; APC, adenomatous polyposis coli; ASPM, abnormal spindle microtubule assembly; AURKA, aurora kinase A; CDC7, cell division cycle 7; CUL3, Cullin 3; DDX50, DExD-box helicase 50; DYNC1I2, dynein cytoplasmic 1 intermediate chain 2; ESR1, estrogen receptor 1; HIST1H4C, histone cluster 1 H4 family Member C; HTATSF1, HIV-1 tat specific factor 1; KIF23, kinesin family member 23; LIG1, DNA ligase 1; KCTD6, potassium channel tetramerization domain containing 6; MRPS9, mitochondrial ribosomal protein S9; NT, nontumor; NUP205, nucleoporin 205; OIP5, Opa interacting protein 5; PRIM1, DNA primase subunit 1; PSPC1, paraspeckle component 1; PWP1, PWP1 homolog, endonuclein; RB1, RB transcriptional corepressor 1; RBM, RNA binding motif protein; RPL, ribosomal protein L; RPS12, ribosomal protein S12; RRBP1, ribosome binding protein 1; SARS2, seryl-TRNA synthetase 2; SEC31A, SEC31 homolog A, COPII Coat Complex component; SNRPB, small nuclear ribonucleoprotein polypeptides B and B1; T, tumor; TECR, trans -2,3-enoyl-CoA reductase; TTC5, tetratricopeptide repeat domain 5; WDR18, WD repeat domain 18; ZNF320, zinc finger protein 320.

Article Snippet: The human Genome-Scale CRISPR Knock-Out (GeCKO) v2 CRISPR knockout pooled library was a gift from Feng Zhang (Broad Institute, Massachusetts Institute of Technology, Cambridge, MA, USA) (Addgene 1000000048).

Techniques: CRISPR, Microarray, RNA Binding Assay, Binding Assay