Journal: Scientific Data

Article Title: Comprehensive genome-scale CRISPR knockout screening of CHO cells

doi: 10.1038/s41597-025-04438-6

Figure Lengend Snippet: Schematic illustration of a non-viral, genome-scale CRISPR screening platform in Chinese hamster ovary (CHO) cells. ( a ) Plasmid library construction. CHO genome-wide CRISPR guide RNA (gRNA) oligonucleotides were designed and synthesized. Recombinase mediated cassette exchange (RMCE) donor plasmid library was then constructed by cloning gRNA oligos into a backbone plasmid that contained a promoter-less puromycin resistance (PuroR) gene and gRNA scaffold, flanked by attB and mutant attB recombination sites. The plasmid library was verified using next-generation sequencing (NGS) analysis. ( b ) Workflow of cell library and knockout (KO) library development. The CHO-K1 host and recombinant cell-based master cell lines (MCLs) were established through CRISPR/Cas9-mediated site-specific integration (SSI) of the landing pad (LP) plasmid donor, which contained mCherry and hygromycin resistance (HygR) gene flanked by attP and mutant attP recombination sites. The host and recombinant cell libraries were generated through the Bxb1-att recombination system and puromycin selection. Promoter-trapping ensures the expression of a single gRNA per cell. The host and recombinant KO libraries were then generated through transient Cas9 expression and blasticidin semi-selection. The distribution of gRNAs in the pooled cell libraries and KO libraries was confirmed using NGS analysis. The KO libraries were also analyzed after long-term cultivation. ( c ) Functional genomic screens. Phenotypes of interest in the KO library can be enriched through reporter-based fluorescence-activated cell sorting (FACS) or pool selection under selective pressure. Highly productive recombinant cell populations were sorted in this study. The distribution of gRNAs was confirmed using NGS analysis. The screening hits are candidate targets for KO cell engineering.

Article Snippet: CRISPR screening data analysis is available at https://sourceforge.net/p/mageck/wiki/Home/#usage and http://pinapl-py.ucsd.edu .

Techniques: CRISPR, Plasmid Preparation, Genome Wide, Synthesized, Construct, Cloning, Mutagenesis, Next-Generation Sequencing, Knock-Out, Recombinant, Generated, Selection, Expressing, Functional Assay, Fluorescence, FACS

Journal: Scientific Data

Article Title: Comprehensive genome-scale CRISPR knockout screening of CHO cells

doi: 10.1038/s41597-025-04438-6

Figure Lengend Snippet: The development process of the cell and KO libraries using a non-viral genome-scale CRISPR screening platform. Entire profiles of ( a ) viability and ( b ) cell growth (VCD; viable cell density) during the generation of the cell (red solid lines) and KO (blue solid lines) libraries in both CHO-K1 host and recombinant cells. Arrows indicate the transfection of RMCE (red) and Cas9 (blue). Shaded areas denote periods of chemical treatment for cell library enrichment (red, puromycin) and KO library enrichment (blue, blasticidin). The pEGFP-c1 expression vector was used as the transfection control (green lines), while chemically untreated cells served as non-enriched controls (dotted lines). ( c ) The percentage of mCherry-negative populations in the CHO-K1 host (blue line) and recombinant cells (red line) during cell library development with and without puromycin selection. Dotted line indicates the threshold required to maintain 500 × gRNA coverage. The mCherry negativity in enriched populations indicated successful implementation of the genome-scale plasmid library into cells. ( d ) Bar plot showing the transfection efficiency of Cas9 in CHO-K1 host cells and recombinant cells as measured through 2 A peptide-linked reporter expression.

Article Snippet: CRISPR screening data analysis is available at https://sourceforge.net/p/mageck/wiki/Home/#usage and http://pinapl-py.ucsd.edu .

Techniques: CRISPR, Recombinant, Transfection, Expressing, Plasmid Preparation, Control, Selection

Journal: Cancers

Article Title: MiRNA Deregulation Distinguishes Anaplastic Thyroid Carcinoma (ATC) and Supports Upregulation of Oncogene Expression

doi: 10.3390/cancers13235913

Figure Lengend Snippet: Unsupervised analyses of small RNA expression data from 10 ATCs, 6 PTCs, 6 FTCs, and 6 nonmalignant human thyroid (NT, normal thyroid) samples of 85 miRNAs with significantly deregulated expression (DE) in ATC. ( A ) Principal component analysis (PCA) shows that DE miRNAs distinguish ATC from all other samples analyzed, except for ATC#10. ( B ) Hierarchical clustering of the 85 significantly deregulated miRNAs supports the PCA analyses and sharply distinguishes all ATC samples, except ATC#10. Rows are ordered by average log2 fold change of miRNA expression in ATC samples compared to all other samples analyzed. Normalized log2 CPM (counts per million mapped reads) expression values of identified miRNAs were used for both analyses.

Article Snippet: To integrate essentiality information of the determined miRNA target genes in ATC, pancancer loss-of-function CRISPR screens of nine ATC-derived cell lines (8305C, 8505C, MB1, ASH3, BHT101, CAL62, HOTHC, HTCC3, KMHDASH2) were utilized for a dependency analysis, using the Broad Institute Cancer Dependency Map (DepMap) portal (v 21Q1, [ ]).

Techniques: RNA Expression, Expressing

Journal: Cancers

Article Title: MiRNA Deregulation Distinguishes Anaplastic Thyroid Carcinoma (ATC) and Supports Upregulation of Oncogene Expression

doi: 10.3390/cancers13235913

Figure Lengend Snippet: The established analysis pipeline showing the rational for selection of prime miRNA–mRNA interactions in ATC. In brief, the miRNA and mRNA expression were determined by RNA-seq in indicated samples. Differential miRNA expression identified 85 differentially expressed (DE) miRNAs at the threshold of FDR < 0.01 and an average expression > 100 CPM. To identify key effector mRNAs of DE miRNAs, miRNA targeting was analyzed by in silico prediction using indicated databases. Candidate miRNA–mRNA interactions were further ranked by the fold change (FC) of target mRNA expression, average essentially scores (ES) were determined in ATC derived cells for target genes, and hazard ratios (HR) were determined by target mRNA expression in the TCGA thyroid cancer cohort (THCA). This led to an interaction score (IS) for each miRNA-mRNA interaction scaled as indicated. Finally, prime candidate oncogenic and tumor-suppressive target mRNAs were determined for the top scoring (IS = 1) target mRNA of each DE miRNA by the indicated parameters. This allowed a ranking of miRNA-controlled candidate oncogenic and tumor-suppressive factors via a scaled OncoScore (OS), as indicated. Bars indicate a scale of 100 µm.

Article Snippet: To integrate essentiality information of the determined miRNA target genes in ATC, pancancer loss-of-function CRISPR screens of nine ATC-derived cell lines (8305C, 8505C, MB1, ASH3, BHT101, CAL62, HOTHC, HTCC3, KMHDASH2) were utilized for a dependency analysis, using the Broad Institute Cancer Dependency Map (DepMap) portal (v 21Q1, [ ]).

Techniques: Selection, Expressing, RNA Sequencing, In Silico, Derivative Assay

Journal: Cancers

Article Title: MiRNA Deregulation Distinguishes Anaplastic Thyroid Carcinoma (ATC) and Supports Upregulation of Oncogene Expression

doi: 10.3390/cancers13235913

Figure Lengend Snippet: Gene Set Enrichment Analysis (GSEA) of total and miRNA-dependent differential mRNA expression in ATC. Columns indicate GSEA results using distinct mRNA selection: FC mRNA, all expressed mRNAs were ranked by their log fold change of expression in ATC compared to all other samples; DN miRNA, candidate target mRNAs with positive interaction scores (IS) determined for DN-miRNAs were ranked by their log fold change of expression in ATC compared to all other samples; UP miRNA candidate target mRNAs with positive interaction scores (IS) determined for UP-miRNAs also ranked by their log fold change of expression in ATC. Color coding indicates scaled normalized enrichment scores (NES) determined for indicated hallmark gene sets. NES scale bar is indicated.

Article Snippet: To integrate essentiality information of the determined miRNA target genes in ATC, pancancer loss-of-function CRISPR screens of nine ATC-derived cell lines (8305C, 8505C, MB1, ASH3, BHT101, CAL62, HOTHC, HTCC3, KMHDASH2) were utilized for a dependency analysis, using the Broad Institute Cancer Dependency Map (DepMap) portal (v 21Q1, [ ]).

Techniques: Expressing, Selection

Journal: Cancers

Article Title: MiRNA Deregulation Distinguishes Anaplastic Thyroid Carcinoma (ATC) and Supports Upregulation of Oncogene Expression

doi: 10.3390/cancers13235913

Figure Lengend Snippet: High ranking miRNA–mRNA interactions identify miRNA-dependent regulation of oncogenic and tumor-suppressive factors in ATC. ( A ) Hierarchical clustering of the top scoring (IS = 1) target mRNAs of the 85 DE miRNAs sharply distinguish all ATC samples. Samples were ordered by the scaled OncoScore (OS), indicating the oncogenic (top score 1) vs. tumor-suppressive (top score −1) potential of the respective genes. Gene symbols are indicated in the left panel. Log2 transformed FPKM expression values of indicated mRNAs scaled to low and high expression range [−1, 1] are depicted by a heatmap in the middle panel. Prime targeting miRNAs of indicated mRNAs are indicated in the right panel along with the scaled [−1, 1] fold change of miRNA expression in ATC vs. others. ( B , C ) Expression and miRNA targeting of the prime oncogene upregulated by miRNA downregulation (BIRC5, ( B )) and the prime tumor-suppressor candidate decreased by miRNA upregulation (FRMD3, ( C )). Boxplots (left panels) show BIRC5 and FRMD3 mRNA (log2 FPKM) expression in ATC and others; **** p < 0.0001. Schemes (middle panels) depict the meta 3′-UTR of selected genes with predicted binding sites for targeting miRNAs with average expression >100 CPM. Fold change of miRNA expression is indicated by up/down bars scaled by fold change of expression. Color coding indicates significant deregulation (FDR < 0.01). Nonsignificant in grey. The sum CPM of targeting miRNAs sorted by significant (color coded) or insignificant change of expression (grey) is indicated in the right panels.

Article Snippet: To integrate essentiality information of the determined miRNA target genes in ATC, pancancer loss-of-function CRISPR screens of nine ATC-derived cell lines (8305C, 8505C, MB1, ASH3, BHT101, CAL62, HOTHC, HTCC3, KMHDASH2) were utilized for a dependency analysis, using the Broad Institute Cancer Dependency Map (DepMap) portal (v 21Q1, [ ]).

Techniques: Transformation Assay, Expressing, Binding Assay

Journal: Cancers

Article Title: MiRNA Deregulation Distinguishes Anaplastic Thyroid Carcinoma (ATC) and Supports Upregulation of Oncogene Expression

doi: 10.3390/cancers13235913

Figure Lengend Snippet: Expression of miRNA-dependent oncogenic factors DTL and TFRC distinguishes ATC. ( A ) Expression of DTL and TFRC analyzed by immunohistochemistry (IHC) in representative samples derived from a tissue microarray (TMA). H&E, hematoxylin eosin staining; magnification 1:40; bars indicate a scale of 50 µm. ( B , C ) Evaluation of IHC samples by histoscore (H-score). Violin plots show the H-score distributions determined for DTL ( B ) and TFRC ( C ) in indicated samples. ANOVA testing confirms significantly distinct expression. Gene specific OncoScores (OS) are depicted in top panels.

Article Snippet: To integrate essentiality information of the determined miRNA target genes in ATC, pancancer loss-of-function CRISPR screens of nine ATC-derived cell lines (8305C, 8505C, MB1, ASH3, BHT101, CAL62, HOTHC, HTCC3, KMHDASH2) were utilized for a dependency analysis, using the Broad Institute Cancer Dependency Map (DepMap) portal (v 21Q1, [ ]).

Techniques: Expressing, Immunohistochemistry, Derivative Assay, Microarray, Staining

Journal: Nature

Article Title: BRD8 maintains glioblastoma by epigenetic reprogramming of the p53 network

doi: 10.1038/s41586-022-05551-x

Figure Lengend Snippet: a, Pie charts showing frequency of TP53 mutation in cBioPortal GBM patient cohorts. b, Kaplan-Meier survival curve showing overall survival after first diagnosis of GBM patients with or without TP53 mutation. P value was calculated by Log-rank (Mantel-Cox) test. c, Systematic statistics of p53 mutations across all p53 codons, with the x-axis showing p53 codons with annotation of conserved domains across the protein shown below, and the y-axis showing the frequency of mutation at each codon. TP53 mutation data was compiled from the TCGA database and previous publications. d and e, RT-qPCR showing CDKN1A expression in the GBM cell lines used for CRISPR screens (d) and the primary patient-derived xenograft GBM cells used for validation (e), as compared with non-malignant human brain-derived neural stem cells (BNSC). Plotted is the relative expression level normalized to GAPDH as the mean ± s.d. (n = 3 biologically independent samples). f and g, MTT-based proliferation assays in the indicated GBM cell lines (f), primary patient cells and xenograft cells (g). Cells were treated with the MDM2 inhibitor Nutlin-3a, and were subjected to MTT assays after three population doublings (normalized to DMSO, n = 3 biologically independent samples). P values were calculated using two-tailed unpaired Student’s t-tests. h, Work flow of CRISPR screens. i, Comparison of domain-focused CRISPR screen in this study with genome-wide CRISPR screen data from the DepMap (https://depmap.org/portal/) using the same cell lines across diverse cancer types.

Article Snippet: Anexon-tiling CRISPR screen used tofunctionally scan the entire protein also revealed that the bromodomain was the most important module within BRD8 in TP53 WT GBM ( ).

Techniques: CRISPR, Mutagenesis, Biomarker Discovery, Quantitative RT-PCR, Expressing, Derivative Assay, Two Tailed Test, Comparison, Genome Wide

Journal: Nature

Article Title: BRD8 maintains glioblastoma by epigenetic reprogramming of the p53 network

doi: 10.1038/s41586-022-05551-x

Figure Lengend Snippet: a, Summary of the sgRNAs used in this study targeting BRD8 exons encoding the bromodomain (sgBRD8–1 and sgBRD8–2) or introns (sgIntron-1 and sgIntron2) flanking the exons encoding the bromodomain. b, Western blots showing BRD8 depletion by two independent sgRNAs in the indicated cells. Data shown represents two independent results. sgNeg is a negative control. HSC70 serves as loading control. c, Cellular competition-based GFP dropout assays of individual sgRNAs targeting BRD8 and MDM2 along with sgNeg and a positive control sgRNA targeting the replication protein CDK1 (sgCDK1). Plotted is the percentage of independent GFP positive cells (normalized to P0, n = 3 biologically independent samples) at the indicated time points. P0 refers to day 3 after infection. d, Western blot showing BRD8 levels after targeting its introns by two independent sgRNAs (sgIntron-1 and sgIntron-2) (top) and GFP dropout assays along with sgNeg and sgCDK1 in A382WT cells (bottom). Plotted is the mean ± s.d. (normalized to P0, n = 3 biologically independent samples). e, Western blots showing BRD8 knockdown in A382WT cells transduced with two individual shRNAs targeting BRD8 (shBRD8–1 and shBRD8–2) (top) and GFP dropout assays in A382WT with shRluc as a negative control and shRPA3 as a positive control (bottom). Plotted is the mean ± s.d. (normalized to P0, n = 3 biologically independent samples). f, Design of CRISPR-resistant BRD8 cDNAs against sgBRD8–1 (BRD8 CR-1) or sgBRD8–2 (BRD8 CR-2). g, Western blots showing overexpression of CRISPR-resistant BRD8 cDNAs (BRD8 CR-1 and BRD8 CR-2) in the two indicated TP53WT GBM cells with empty vector as control (Ctrl). h and i, GFP dropout assays of sgNeg, sgCDK1, sgBRD8–1 and sgBRD8–2 in indicated TP53WT GBM cells expressing Ctrl or CRISPR-resistant BRD8 cDNAs (BRD8 CR-1 or BRD8 CR-2). Plotted is the mean ± s.d. (normalized to P0, n = 3 biologically independent samples). j, MTT-based proliferation assays in primary GBM patient cells, xenograft cells, and immortalized brain-derived neural stem cells (BNSC). Plotted is the mean ± s.d. (normalized to sgNeg, n = 3 biologically independent samples). P values were calculated using two-tailed unpaired Student’s t-tests. k, The top panel depicts the Aka luciferase-expressing construct used in orthotopic brain transplantation assays for noninvasive imaging in vivo. Bottom left: in vitro validation of luciferase activity in cultured cells using D-luc or AkaLuc-HCL substrates in empty control (Ctrl), Fluc-expressing (Fluc), and AkaLuc-expressing (AkaLuc) cells. Bottom right: Bioluminescence imaging of NOD SCID mice that had orthotopic brain transplantation of AkaLuc-expressing U87WT cells transduced with sgNeg or sgBRD8–2 (see correlating Kaplan-Meier survival plot, Fig. 1b). l and m, Kaplan-Meier survival plot of recipient mice that had been orthotopically transplanted with A382WT (l) or U251R273H (m) GBM cells expressing sgNeg or sgBRD8 into the brains of recipient mice. P value was calculated by Log-rank (Mantel-Cox) test.

Article Snippet: Anexon-tiling CRISPR screen used tofunctionally scan the entire protein also revealed that the bromodomain was the most important module within BRD8 in TP53 WT GBM ( ).

Techniques: Western Blot, Negative Control, Control, Positive Control, Infection, Knockdown, Transduction, CRISPR, Over Expression, Plasmid Preparation, Expressing, Derivative Assay, Two Tailed Test, Luciferase, Construct, Transplantation Assay, Imaging, In Vivo, In Vitro, Biomarker Discovery, Activity Assay, Cell Culture

Journal: Nature

Article Title: BRD8 maintains glioblastoma by epigenetic reprogramming of the p53 network

doi: 10.1038/s41586-022-05551-x

Figure Lengend Snippet: a, Heat map showing CRISPR screen results for the entire family of bromodomain-containing proteins (classified into eight subgroups) in the indicated cancer types. b, Construction of the CRISPR resistant BRD8 cDNA against sgBRD8–2 tagged with 3XFlag (CR-2-C3F) and the bromodomain-deleted BRD8 cDNA tagged with 3XFlag (BDdel-C3F) (left). Expression is validated using western blot in A382WT cells (right). Data shown represents two independent results. HSC70 is a loading control. c, Confocal microscopy showing nuclear localization of both wild type (BRD8-C3F) and bromodomain deleted BRD8 (BDdel-C3F). Scale bar, 50 μm. Data shown represents three independent results.

Article Snippet: Anexon-tiling CRISPR screen used tofunctionally scan the entire protein also revealed that the bromodomain was the most important module within BRD8 in TP53 WT GBM ( ).

Techniques: CRISPR, Expressing, Western Blot, Control, Confocal Microscopy