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 Communications

Article Title: Identification of regulators of poly-ADP-ribose polymerase inhibitor response through complementary CRISPR knockout and activation screens

doi: 10.1038/s41467-020-19961-w

Figure Lengend Snippet: a Schematic representation of the CRISPR knockout screen for olaparib sensitivity in wildtype cells. HeLa cells were infected with the Brunello CRISPR knockout library. Infected cells were divided into PARP inhibitor (olaparib)-treated or control (DMSO) arms. Genomic DNA was extracted from cells surviving the drug treatment and single-guide RNAs (sgRNAs) were identified using Illumina sequencing. b Scatterplot showing the results of this screen. Each gene targeted by the library was ranked based on the MAGeCK negative selection score. Several biologically interesting hits are highlighted. c Schematic representation of the CRISPR knockout screen for olaparib resistance in BRCA2 KO cells. HeLa BRCA2 KO cells were infected with the Brunello CRISPR knockout library. Infected cells were divided into PARP inhibitor (olaparib)-treated or control (DMSO) arms. d Scatterplot showing the results of this screen, with several biologically interesting hits highlighted. Each gene targeted by the library was ranked based on the MAGeCK positive selection score. e Schematic representation of the CRISPR activation screen for olaparib resistance in BRCA2 KO cells. HeLa BRCA2 KO cells stably expressing the modified dCas9 enzyme were infected with the Calabrese CRISPR activation library. Infected cells were divided into PARP inhibitor (olaparib)-treated or control (DMSO) arms. f Scatterplot showing the results of this screen, with several biologically interesting hits highlighted. Each gene targeted by the library was ranked based on the MAGeCK positive selection score. Source data are provided as a Source Data file.

Article Snippet: For the CRISPR activation screens, HeLa BRCA2-knockout cells were infected with dCas9 (Addgene, 61425-LV) and selected with blasticidin (3 μg/ml). dCas9-expressing cells were then transduced with the Calabrese Human CRISPR Activation Pooled Library (Set A, Addgene, 92379-LV) using enough cells to obtain a library coverage of 500 cells per sgRNA at an MOI of 0.4 .

Techniques: CRISPR, Knock-Out, Infection, Control, Illumina Sequencing, Selection, Activation Assay, Stable Transfection, Expressing, Modification

Journal: Nature Communications

Article Title: Identification of regulators of poly-ADP-ribose polymerase inhibitor response through complementary CRISPR knockout and activation screens

doi: 10.1038/s41467-020-19961-w

Figure Lengend Snippet: a Western blot showing overexpression of ABCB1 in the cell line containing all three components of the CRISPR lentiviral activation particle (LAP) system (dCas9, activator helper complex, and sgRNA targeting the ABCB1 gene) but not in the control cell line lacking the sgRNA. b Cellular viability assay showing that ABCB1 transcriptional activation rescues PARPi sensitivity in HeLa BRCA2-knockout cells. The averages of four experiments are shown, with standard deviations as error bars. c Olaparib-induced apoptosis in BRCA2-knockout cells is suppressed by ABCB1 overexpression. The averages of four experiments are shown, with standard deviations as error bars. Asterisks indicate statistical significance ( t -test, two-tailed, unequal variance) compared to the No Guide sample. Source data are provided as a Source Data file.

Article Snippet: For the CRISPR activation screens, HeLa BRCA2-knockout cells were infected with dCas9 (Addgene, 61425-LV) and selected with blasticidin (3 μg/ml). dCas9-expressing cells were then transduced with the Calabrese Human CRISPR Activation Pooled Library (Set A, Addgene, 92379-LV) using enough cells to obtain a library coverage of 500 cells per sgRNA at an MOI of 0.4 .

Techniques: Western Blot, Over Expression, CRISPR, Activation Assay, Control, Cell Viability Assay, Knock-Out, Two Tailed Test

Journal: Cell Reports

Article Title: Quantitative Proteomics Analysis of Lytic KSHV Infection in Human Endothelial Cells Reveals Targets of Viral Immune Modulation

doi: 10.1016/j.celrep.2020.108249

Figure Lengend Snippet: KSHV CRISPR Library Screen Identifies K5 as ORF Responsible for Downregulation of the NK Cell Receptor Ligands Nectin-2 and CD155 (A) Immunoblot analysis of HuAR2T.rKSHV.219 Cas9 cells harboring sgRNAs specific for indicated viral ORFs untreated or treated with reactivation mix and probed with the indicated antibodies. A non-specific band is labeled with an asterisk. (B) Schematic flowchart of the genetic screen with the KSHV CRISPR library. HuAR2T.rKSHV.219 Cas9 cells were transduced with the sgRNA library followed by lytic KSHV cycle induction and stained with CD155- or Nectin-2-specific antibody. Lytic (RFP+) and CD155- or Nectin-2-high cells were selected by FACS and used for DNA extraction and sequencing. (C and D) Genetic screens with the KSHV CRISPR library identify K5 as the ORF responsible for downregulation of Nectin-2 and CD155. Each dot represents a single KSHV ORF or miRNA plotted by the statistical significance (-log p value) of the sgRNA enrichment. (E) CRISPR-mediated K5 knockout leads to full (Nectin-2) or partial (CD155) rescue of lytic KSHV-induced downregulation of the proteins from the cell surface. Flow cytometry analysis of HuAR2T.rKSHV.219 Cas9 cells harboring control or K5-specific sgRNAs untreated or treated with reactivation mix and stained with the indicated antibody. (F) Flow cytometry analysis of HuAR2T cells transduced with LV K5 or control LV and probed with the indicated antibody.

Article Snippet: The resulting library contained 1281 KSHV-specific and 50 non-targeting sgRNAs from the Activity-Optimized Human CRISPR Pooled Library (Addgene #1000000067) ( ) (sequences are presented in ). sgRNA sequences flanked by BsmBI restriction sites and unique adapters ( ) were synthesized as a pool of single-stranded oligonucleotides by Custom Array (Bothell, USA).

Techniques: CRISPR, Western Blot, Labeling, Transduction, Staining, DNA Extraction, Sequencing, Knock-Out, Flow Cytometry, Control

Journal: Cell Reports

Article Title: Quantitative Proteomics Analysis of Lytic KSHV Infection in Human Endothelial Cells Reveals Targets of Viral Immune Modulation

doi: 10.1016/j.celrep.2020.108249

Figure Lengend Snippet: CRISPR Proteomics Identifies 48 KSHV K5 Targets in Endothelial Cells (A) Schematic overview of the quantitative proteomic analysis of cells with lytic versus latent KSHV infection. HuAR2T.rKSHV.219 Cas9 cells harboring control or K5-specific sgRNAs were transduced with LV RTA or control LV BFP, sorted on BFP+ or RFP+, and analyzed by MS. (B) KSHV K5 protein significantly (q < 0.1) downregulates 48 proteins in endothelial cells, of which 30 are known K5 targets (left side of blue circle) and 18 proteins represent K5 targets identified in this study (yellow circle). (C–E) Scatterplots display pairwise comparison between lytic control and CRISPR K5 (C), latent and lytic control (D), and latent and lytic CRISPR K5 (E) KSHV infections. Each point represents a single protein, plotted by its log2 (fold change in abundance) versus the statistical significance (p and q value) of that change. Values were corrected for multiple hypothesis testing using the method of Benjamini-Hochberg. Dotted lines: q = 0.05. (F) Flow cytometry analysis of HuAR2T cells transduced with LV K5 or control LV and stained with isotype control antibody or antibody specific for the indicated proteins. (G and H) Immunoblot analysis of HuAR2T cells transduced with LV K5 or control LV, sorted on GFP+, and probed with the indicated antibody. See also Figure S2 and .

Article Snippet: The resulting library contained 1281 KSHV-specific and 50 non-targeting sgRNAs from the Activity-Optimized Human CRISPR Pooled Library (Addgene #1000000067) ( ) (sequences are presented in ). sgRNA sequences flanked by BsmBI restriction sites and unique adapters ( ) were synthesized as a pool of single-stranded oligonucleotides by Custom Array (Bothell, USA).

Techniques: CRISPR, Infection, Control, Transduction, Comparison, Flow Cytometry, Staining, Western Blot

Journal: Cell Reports

Article Title: Quantitative Proteomics Analysis of Lytic KSHV Infection in Human Endothelial Cells Reveals Targets of Viral Immune Modulation

doi: 10.1016/j.celrep.2020.108249

Figure Lengend Snippet: DAVID GO Term Analysis of Proteins Dysregulated by Lytic KSHV Infection (A) Ten most enriched GO terms ranked by statistical significance (p value) in the category “molecular function” among proteins upregulated by lytic KSHV. (B) Histogram shows the fold change in the abundance of the proteins from the GO term “unfolded protein binding.” (C) Upregulated proteins (red points) from the GO term “poly(A) RNA binding” are highlighted on the scatterplot that displays pairwise comparison between latent and lytic KSHV infections. Each point represents a single protein, plotted by its log2 (fold change in abundance) versus statistical significance (q value) of that change. Value was corrected for multiple hypothesis testing using the method of Benjamini-Hochberg. Dotted line: q = 0.05 (D) Ten most enriched GO terms ranked by statistical significance (p value) in the category “molecular function” among proteins downregulated by lytic KSHV. (E) Validation of lytic KSHV-mediated downregulation of the surface proteins from the GO terms “integrin binding” and “transmembrane receptor protein tyrosine kinase activity.” (F) Relative abundance of the indicated host proteins in the cells with latent (gray), lytic CRISPR control (blue) and lytic CRISPR K5 (green) KSHV infections. Protein abundance is calculated as a fraction of the maximum TMT reporter ion intensity. Data are represented as mean ± SEM. (G–J) Downregulated proteins from the GO terms “protein kinase activity” (G) and “chromatin binding” (I) are highlighted on the scatterplots that display pairwise comparison between latent and lytic KSHV infection. Each point represents a single protein, plotted by its log2 (fold change in abundance) versus statistical significance (q value) of that change. Values were corrected for multiple hypothesis testing using the method of Benjamini-Hochberg. Dotted line: q = 0.05. (H and J) Immunoblot analysis of HuAR2T.rKSHV.219 cells transduced with LV RTA or control LV BFP, sorted on BFP+ or RFP+, and probed with the indicated antibody. Non-specific bands are labeled with an asterisk. See also Figure S5 and .

Article Snippet: The resulting library contained 1281 KSHV-specific and 50 non-targeting sgRNAs from the Activity-Optimized Human CRISPR Pooled Library (Addgene #1000000067) ( ) (sequences are presented in ). sgRNA sequences flanked by BsmBI restriction sites and unique adapters ( ) were synthesized as a pool of single-stranded oligonucleotides by Custom Array (Bothell, USA).

Techniques: Infection, Protein Binding, RNA Binding Assay, Comparison, Biomarker Discovery, Binding Assay, Activity Assay, CRISPR, Control, Quantitative Proteomics, Western Blot, Transduction, Labeling

Journal: Cell Reports

Article Title: Quantitative Proteomics Analysis of Lytic KSHV Infection in Human Endothelial Cells Reveals Targets of Viral Immune Modulation

doi: 10.1016/j.celrep.2020.108249

Figure Lengend Snippet:

Article Snippet: The resulting library contained 1281 KSHV-specific and 50 non-targeting sgRNAs from the Activity-Optimized Human CRISPR Pooled Library (Addgene #1000000067) ( ) (sequences are presented in ). sgRNA sequences flanked by BsmBI restriction sites and unique adapters ( ) were synthesized as a pool of single-stranded oligonucleotides by Custom Array (Bothell, USA).

Techniques: Purification, Control, Virus, Recombinant, Mass Spectrometry, Sample Prep, Transfection, SYBR Green Assay, Sequencing, CRISPR, Plasmid Preparation, Software