Journal: Cell systems

Article Title: Illuminating Host-Mycobacterial Interactions with Genome-wide CRISPR Knockout and CRISPRi Screens.

doi: 10.1016/j.cels.2020.08.010

Figure Lengend Snippet: Figure 1. A Pooled Approach for CRISPR Knockout and CRISPRi Screening in Human THP-1 Cells (A) Strategy for preparing CRISPR libraries and performing genetic screens. (B) THP-1-mediated phagocytosis of M. bovis BCG after three rounds of infection (MOI 10:1) with induced green fluorescence (map24::GFP) (Scale bar, 20 mm). (C) Viability of host cells after three rounds of M. bovis BCG infection. (D and E) Expression of Cas9 (D) and dCas9-KRAB (E) in 9 randomly selected monoclonal THP-1 cells. Wild-type THP-1 cells were used as negative control. Vinculin was used as a loading control. (F) An sgRNA for EGFP was introduced in both wild-type and Cas9-expressing THP-1 cells using a lentivirus (pXPR-011) that also contains EGFP as a target (Scale bar, 20 mm). (G) Cas9-expressing THP-1 cells were transduced with an sgRNA targeting AAVS1 at a low MOI. Mutations at the AAVS1 locus were detected by SURVEYOR assay. The size of the AAVS1 amplicon is 500 bp. The cleaved product sizes are 320 and 180 bp. (H) Growth measurement associated with sgRNAs targeting INTS9, MCM2, and non-targeting negative controls sgNC1 and sgNC13. (I and J) RT-qPCR analysis of INTS9 (I) and MCM2 (J) expression in dCas9-KRAB-expressing THP-1 cells. The values are normalized to GAPDH (glyceraldehyde- 3-phosphate dehydrogenase). Data represent the mean ± SD (n = 3) (two-tailed unpaired Student’s t test, *p < 0.05 **p < 0.01 ***p < 0.001). See also Figure S1; Table S13.

Article Snippet: Human CRISPR knockout pooled library (Brunello) was obtained from Addgene (#73178).

Techniques: CRISPR, Knock-Out, Infection, Expressing, Negative Control, Control, Transduction, Amplification, Quantitative RT-PCR, Two Tailed Test

Journal: Cell systems

Article Title: Illuminating Host-Mycobacterial Interactions with Genome-wide CRISPR Knockout and CRISPRi Screens.

doi: 10.1016/j.cels.2020.08.010

Figure Lengend Snippet: Figure 2. Genome-wide Pooled CRISPR Knockout and CRISPRi Screens to Dissect Biological Pathways in Mycobacterial Infection (A and B) Volcano plots from CRISPR knockout (A) and CRISPRi (B) screens. For each sgRNA-targeted gene, the x axis shows its enrichment or depletion post- infection, and the y axis shows statistical significance measured by p value. Positive and negative screen hits are labeled as red and green dots, respectively. Gray dots represent non-targeting controls. For each screen, experiments were carried out in triplicate. (C) Enriched genes in the Venn diagram were filtered with a cut-off of FDR <0.1 and log2-fold change >1 in M. bovis BCG infection. The degree of significance of the overlap is given. (D) Gene-centric visualization of average fold change of CRISPR knockout and CRISPRi screens in infected versus non-infected host cells. Selected type I IFN and AHR/ARNT pathway components are highlighted in orange and blue. (E and F) Candidate genes identified by CRISPR knockout (E) and CRISPRi (F) screens were functionally categorized to understand the changes in biological functions involved in M. bovis BCG infection. Pathways shown in red are those identified by both screens. Color gradient of nodes represents the enrichment scores of gene sets. Node size represents the number of genes in the gene set. Edge width represents mutual overlap of genes. See also Figures S2 and S3; Tables S1, S2, S3, S4, S5, S11, and S12.

Article Snippet: Human CRISPR knockout pooled library (Brunello) was obtained from Addgene (#73178).

Techniques: Genome Wide, CRISPR, Knock-Out, Infection, Labeling

Journal: Cell systems

Article Title: Illuminating Host-Mycobacterial Interactions with Genome-wide CRISPR Knockout and CRISPRi Screens.

doi: 10.1016/j.cels.2020.08.010

Figure Lengend Snippet: Figure 3. Secondary CRISPR Knockout and CRISPRi Screens Identify Host Genetic Hits in Mycobacterial Infection (A) Enriched genes were filtered with a cut-off of FDR <0.05 and log2-fold change >0.5 in M. bovis BCG infection. The degree of significance of the overlap is given. (B) Validation rate of genetic hits in secondary screens grouped by their p values in primary genome-wide screens in M. bovis BCG infection. Number of genes per category is indicated. (C) Genetic hits from both primary and secondary screens were ranked by their differential sgRNA abundance between M. bovis BCG-infected versus uninfected populations (log2 fold change). (D) Heatmap of screen hits (log2 fold change) clustered in different biological pathways in M. bovis BCG infection. See also Figures S4 and S5; Tables S6, S7, S8, S9, and S10.

Article Snippet: Human CRISPR knockout pooled library (Brunello) was obtained from Addgene (#73178).

Techniques: CRISPR, Knock-Out, Infection, Biomarker Discovery, Genome Wide

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: bioRxiv

Article Title: Parallel CRISPR screens reveal pathways controlling the cell surface levels of the attractant receptor FPR1

doi: 10.1101/2025.04.21.649864

Figure Lengend Snippet: Clonally selected β-arrestin double CRISPR knockout cells are compared to pooled double β-arrestin knockout cell lines using the FPR1 endocytosis assay. Cells were untreated or stimulated with 1 μM fMLF. (A) A representative plot showing fMLF-induced decrease in surface FPR1 in the control sgRNA expressing cells. (B) Pooled double β-arrestin knockout cells have a smaller shift after fMLF stimulation. (C) Representative plots obtained from twelve clonally selected β-arrestin double knockout cell lines. Phenotypes observed in clonally selected and pooled knockout cell lines resemble each other.

Article Snippet: Bassik Lab Human CRISPR Deletion Library , Addgene catalog #101926- 101934, was packaged into lentiviral particles using TransIT-2020 transfection reagent (VWR, catalog #10767-014).

Techniques: CRISPR, Knock-Out, Endocytosis Assay, Control, Expressing, Double Knockout

Journal: bioRxiv

Article Title: Parallel CRISPR screens reveal pathways controlling the cell surface levels of the attractant receptor FPR1

doi: 10.1101/2025.04.21.649864

Figure Lengend Snippet: (A) Schematic representation of a selection of hits found in the parallel whole-genome CRISPR screens. Genes shown are significant hits in at least one screen, and they were organized based on prior literature. The effect scores for each gene are represented as colored rectangles. The scores are only shown if the gene was a hit in the particular screen (pFDR< 0.05). Negative scores indicate a decrease in surface FPR1, while positive scores indicate an increase. For hits regulating FPR1 surface expression post-stimulation (shown in pink and green), the effect scores obtained from the integrated analysis of the two screens were presented. (B) Top hits from the surface FPR1 expression screen. FPR2 and FPR3 were shown for comparison and are not identified as hits in either of the screens. (C) Significant hits from the integrated analysis of FPR1 internalization, recycling, or exocytosis. Scatter plots in B and C represent the same dataset (log Pscore, with the basal surface expression score on the y-axis and post-stimulation score on the x-axis) with different genes highlighted.

Article Snippet: Bassik Lab Human CRISPR Deletion Library , Addgene catalog #101926- 101934, was packaged into lentiviral particles using TransIT-2020 transfection reagent (VWR, catalog #10767-014).

Techniques: Selection, CRISPR, Expressing, Comparison

Journal: Molecular cell

Article Title: CRAMP1 drives linker histone expression to enable Polycomb repression.

doi: 10.1016/j.molcel.2025.05.031

Figure Lengend Snippet: Figure 1. A genome-wide CRISPR-Cas9 genetic screen identifies an essential requirement for CRAMP1 and histone H1.4 in PRC2-mediated reporter repression (A) Schematic representation of GFP reporter repression by the PRC2 complex. (B) The GFP reporter is derepressed upon CRISPR-Cas9-mediated gene disruption of any of the three core PRC2 subunits, as assayed by flow cytometry. (C) A genome-wide CRISPR-Cas9 screen to identify factors required for PRC2 function. Following Cas9 expression in KBM-7 cells harboring the PRC2-sensitive GFP reporter, genome-wide mutagenesis was carried out with the Sabatini/Lander single guide RNA (sgRNA) library, 36 and GFP + cells isolated through two sequential rounds of FACS. ‘‘Significance’’ on the y axis represents the negative log of the ‘‘pos|score’’ metric reported by Model-based Analysis of Genome-wide CRISPR-Cas9 Knockout (MAGeCK). 37

Article Snippet: Single guide RNA (sgRNA) sequences were selected from the Sabatini/Lander Human CRISPR Pooled Library (Addgene #1000000100, kindly deposited by David Sabatini and Eric Lander 81 ) or the Brunello Human CRISPR Knockout Pooled Library (Addgene #73178, kindly deposited by David Root and John Doench 82 ).

Techniques: Genome Wide, CRISPR, Disruption, Flow Cytometry, Expressing, Mutagenesis, Isolation, Knock-Out

Journal: Molecular cell

Article Title: CRAMP1 drives linker histone expression to enable Polycomb repression.

doi: 10.1016/j.molcel.2025.05.031

Figure Lengend Snippet: Figure 5. Linker histones are not enriched at regions marked by H3K9me3 (A–D) Lack of linker histone enrichment at H3K9me3-marked genomic regions. (A) Tornado plots depicting linker histone CUT&Tag signal across H3K9me3 peaks from the ENCODE project; average signal intensity is shown in (B). (C) Heatmap depicting the lack of correlation between linker histone occupancy and H3K9me3. Cells are annotated with pairwise Spearman correlation coefficients. An example locus is shown in (D). (E) CUT&Tag faithfully profiles H3K9me3. Example loci comparing CUT&Tag versus H3K9me3 ChIP-seq data (ENCODE) are shown. (F and G) Linker histone insufficiency does not impair H3K9me3-dependent LINE-1 silencing by the HUSH complex. (F) Schematic representation of the dual- color reporter cell line designed to monitor both H3K9me3-dependent repression by the HUSH complex and linker histone-mediated PRC2-reporter repression. (G) HUSH-mediated LINE-1 silencing is unaffected upon CRAMP1 depletion. The indicated CRISPR sgRNAs were expressed in the dual-color reporter cell line, and GFP and iRFP fluorescence assayed by flow cytometry. See also Figure S5 and Table S2.

Article Snippet: Single guide RNA (sgRNA) sequences were selected from the Sabatini/Lander Human CRISPR Pooled Library (Addgene #1000000100, kindly deposited by David Sabatini and Eric Lander 81 ) or the Brunello Human CRISPR Knockout Pooled Library (Addgene #73178, kindly deposited by David Root and John Doench 82 ).

Techniques: ChIP-sequencing, CRISPR, Fluorescence, Flow Cytometry