Journal: Nature Communications

Article Title: A pan-CRISPR analysis of mammalian cell specificity identifies ultra-compact sgRNA subsets for genome-scale experiments

doi: 10.1038/s41467-022-28045-w

Figure Lengend Snippet: a Schematic illustrating difference between classic synthetic lethality and our common genetic architecture . Synthetic lethality consists of many individual Y i functions. These functions are cell-type-specific models with single features. Our proposed common genetic architecture is hypothesized to connect these “private” functions with shared CERES features. A common genetic architecture has many redundant edges, and more interconnected nodes. More nodes suggest that more cell-type-specific phenotypes are predictable, and more edges suggest redundancy. b A network built from the aggregation of all multivariate models. Genes are represented as nodes and feature-target gene relations as edges. Colors represent distinct subnetwork communities that were identified by the Louvain method. c Network communities with (right) and without (left) nodes/edges involving functional CRISPR features for a single Louvain community, and a comparison with our hypothesis from ( a ). Edges are colored based upon the data source; and nodes are colored based on the model score (of top ten feature model) of the corresponding gene as target. d To quantitate the visual similarity between our hypothesis in ( a ) and the data in ( c ) across all Louvain communities, we examined the differences in the clustering coefficient, the average number of neighbors, and the network heterogeneity. e gprofiler2 plots examine the enrichment of functional categories. f Residual plot identifies GO terms that are more (residuals of −log10 P values >10) or less (residuals of -log10 P values < −10) enriched in predictor genes than in target genes. Dots represent shared GO terms among the 100 most significant terms in target and predictor gprofiler2 analysis result. The p- values from gprofiler2 for ( e ) and ( f ) are based on hypergeometric tests with multiple testing corrections using the g:SCS method. Source data are provided as a Source Data file.

Article Snippet: Human Brunello CRISPR knockout pooled library was a gift from David Root and John Doench (Addgene #73178).

Techniques: Functional Assay, CRISPR, Comparison

Journal: Nature Communications

Article Title: A pan-CRISPR analysis of mammalian cell specificity identifies ultra-compact sgRNA subsets for genome-scale experiments

doi: 10.1038/s41467-022-28045-w

Figure Lengend Snippet: a Two separate pooled screens were performed in a cell line (PC9) that was not included in model training and validation. Experiment 1 was the full Brunello library. A 21-day dropout experiment was performed in PC9 cells. Measurements on 18,114 genes were direct and form the gold standard. The L200 can be computationally extracted from the full screen and compared to these gold-standard measurements. A new L200 standalone library of 800 guides targeting 200 genes was cloned. This library can be used to perform a small-scale lossy compression experiment. The data can then be compared to the gold standard. b Correlations of inferred vs measured CERES scores for both screens in ( a ) and a comparison of the predictions between the standalone sets and the computationally extracted L200 set in the Brunello library. c A Venn diagram describes the overlap in “Hits” in the 500 most differentially required genes for growth in PC9 cells. Both of the lossy compression screens from ( a ) and the gold-standard (measured) data are compared. Source data are provided as a Source Data file.

Article Snippet: Human Brunello CRISPR knockout pooled library was a gift from David Root and John Doench (Addgene #73178).

Techniques: Biomarker Discovery, Clone Assay, Comparison

Journal: Nature Communications

Article Title: CRISPR activation screen identifies BCL-2 proteins and B3GNT2 as drivers of cancer resistance to T cell-mediated cytotoxicity

doi: 10.1038/s41467-022-29205-8

Figure Lengend Snippet: a Schematic of the CRISPRa screen. NY-ESO-1 + and HLA-A2 + A375 melanoma cells were transduced with the pooled sgRNA library targeting more than 23,000 coding isoforms. A375 cells were exposed to primary CD4 + and CD8 + T cells expressing the T cell receptor (TCR) specific for the NY-ESO-1 antigen. Deep sequencing identified candidate genes. b Average MAGeCK analysis P -values for the acute and chronic exposure screens. Top candidate genes are annotated and the two most enriched genes from each screening strategy are highlighted in red. c Most significant pathways enriched among the 576 candidate genes. d Heatmap showing Pearson’s correlation between expression of the top four candidate genes and cytolytic activity across patient tumors from TCGA. Only significant (FDR < 0.05) correlations are shown. e Box plots showing single-sample Gene Set Enrichment Analysis (ssGSEA) of 576 candidate genes in 308 patient tumor samples – . Patient samples were collected prior to treatment with checkpoint inhibitors and classified as responders ( n = 83) or nonresponders ( n = 225) to immunotherapy. Box plots indicate median (middle line), 25th, 75th percentile (box), and 5th and 95th percentile (whiskers). Two-tailed t tests were performed. f Cell survival of A375 cells transduced with ORFs encoding candidate genes against ESO T cell cytotoxicity at different effector to target (E:T) ratios. Cell survival was measured using a luminescent cell viability assay and normalized to paired control cells that were not cultured with T cells. T cells were derived from three donors used in the CRISPRa screen, with n = 4 replicates per donor for n = 12 total. All values are mean ± s.e.m. Two-tailed t tests with adjustments for multiple comparisons were performed. Source data are provided in Source Data 1.

Article Snippet: The human SAM CRISPR activation library (Addgene 1000000057) was used for CRISPR-Cas9 activation screening.

Techniques: Transduction, Expressing, Sequencing, Activity Assay, Two Tailed Test, Cell Viability Assay, Control, Cell Culture, Derivative Assay

Journal: Nature

Article Title: Targeting PIKfyve-driven lipid metabolism in pancreatic cancer.

doi: 10.1038/s41586-025-08917-z

Figure Lengend Snippet: Fig. 3 | PIKfyve inhibition obligates PDAC cells to stimulate a lipogenic transcriptional and metabolic program. a, Schematic of the metabolism- focused CRISPR screen in MIA PaCa-2 cells. Created in BioRender. Cheng, C. (2025) https://BioRender.com/d149928. b,c, Gene enrichment rank plot-based differential sgRNA representation (b) and scatter plot of gene fitness scores (c) of high dose (2,000 nM) and low dose (100 nM) apilimod-treated versus DMSO- treated end-point populations of the CRISPR screen experiment. Graphs show the top 30 synthetically lethal genes involved in fatty acid and sphingolipid synthesis (red), the top 30 synthetically lethal genes involved in cholesterol synthesis (purple) and genes that confer sensitivity to apilimod (blue). d, Metabolic map of sphingolipid and cholesterol synthesis. The figure shows the top 90 synthetically lethal genes involved in fatty acid and sphingolipid

Article Snippet: The human CRISPR metabolic gene knockout library was a gift from David Sabatini (Addgene, 110066)58.

Techniques: Inhibition, CRISPR

Journal: Science (New York, N.Y.)

Article Title: Enhanced T Cell Effector Activity by Targeting Mediator Kinase Module

doi: 10.1126/science.abn5647

Figure Lengend Snippet: A) Schematic depicting CRISPR knockout screen for regulators of cytokine production and CAR-T cell expansion using a tonic signaling model of CAR-T cell exhaustion.

Article Snippet: Cells were maintained at a density between 0.5 and 2 million per mL in T175 flasks. . Lentiviral transduction The complete Bassik Human CRISPR Knockout Library was obtained from Addgene and amplified with Endura ElectroCompetent Cells (Lucigen).

Techniques: CRISPR, Knock-Out

Journal: Nature Communications

Article Title: The CUL5 E3 ligase complex negatively regulates central signaling pathways in CD8 + T cells

doi: 10.1038/s41467-024-44885-0

Figure Lengend Snippet: a Schematic representation of the experimental design for step-wise CRISPR KO screens. The icons are created with BioRender.com. b Scatter plots of sgRNA log 2 -fold change (x-axis, TGFβ1 + IL2/IL2; y-axis, IL2/input) in HT2 cells cultured in vitro for 21 days. c Gene enrichment analysis of the bulk in vivo screen in tumors (Left), spleens (Middle), and TDLNs (Right) using the MAGeCK analysis. d, e Genes enriched in the d , effector cluster and e , proliferating cluster in the single cell CRISPR KO screening are presented as the target gene enrichment over the non-targeting control being plotted against the ratio of the enrichment in d , effector cluster or e , proliferating cluster to that in the exhausted cluster. f Signaling pathways enriched in transferred tumor-infiltrating Cas9/OT-I cells expressing Cul5 sgRNAs compared to those expressing non-targeting sgRNAs.

Article Snippet: The human Brie genome-wide CRISPR knockout pooled library in the pLentiCRISPRv2 one vector system (co-expressing spCas9 and sgRNA), with four sgRNAs per gene, was obtained from Addgene (Addgene # 73632, a gift from David Root and John Doench ) and prepared in the Yale Cancer Center Functional Genomics core). pMSCV-U6sgRNA(BbsI)-PGKpuro2ABFP was a gift from Sarah Teichmann (Addgene plasmid # 102796; http://n2t.net/addgene:102796 ; RRID:Addgene_102796).

Techniques: CRISPR, Cell Culture, In Vitro, In Vivo, Control, Protein-Protein interactions, Expressing

Journal: Genome Research

Article Title: Genome-scale identification of cellular pathways required for cell surface recognition

doi: 10.1101/gr.231183.117

Figure Lengend Snippet: A cell-based genome-scale CRISPR-KO approach identifies pathways required for monoclonal antibody surface epitope recognition. ( A ) Schematic of the approach based on CRISPR/Cas9 technology using a genome-scale lentiviral gRNA library. Genes identified as being required for surface display of the epitope recognized by an anti-CD58 ( B ) and anti-GYPA ( C ) mAbs. The enrichment of gRNAs targeting each gene is quantified as the robust rank aggregation (RRA) score calculated using the MAGeCK software between selected cells that had lost the mAb epitope versus control cells and is shown plotted in rank order. ( D ) Genes identified as being required for surface display of the epitope recognized by an anti-CD59 mAb; here, genes are ordered alphabetically for clarity. Circles represent individual genes and are sized according to their false-discovery rate (FDR): large circle = FDR < 1%, small circle = 1% < FDR < 5%. Genes encoding the direct receptors are indicated with gray circles. Only genes with FDR < 5% are named and are color-coded according to their function.

Article Snippet: The Human Improved Genome-Wide Knockout CRISPR Library v1 (Addgene no. 67989), lentiviral Cas9 reporter plasmids pKLV2-U6gRNA5(gGFP)-PGKBFP2AGFP-W (Addgene no. 67980) and pKLV2-U6gRNA5(Empty)-PGKBFP2AGFP-W (Addgene no. 67979), lentiviral vector expressing Cas9 fused with the blasticidin-resistant gene at the C terminus pKLV2-EF1a-Cas9Bsd-W (Addgene no. 68343), and lentiviral CRISPR gRNA expression vector pKLV2-U6gRNA5(BbsI)-PGKpuro2ABFP-W (Addgene no. 67974) were used in this study.

Techniques: CRISPR, Software, Control

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