Journal: bioRxiv

Article Title: CRlSPR/Cas9 screening revealed BlRC6-AS1 /BlRC6 mediates abiraterone resistance via NHEJ pathway-dependent A20 degradation in prostate cancer

doi: 10.1101/2025.10.01.679907

Figure Lengend Snippet: ( A ) Schematic of CRISPR-Cas9 screens: A lentiviral sgRNA library was transduced into PC3-Cas9 cells, which were then treated with DMSO or Abiraterone, respectively. After 28 days, sgRNAs were extracted for NGS. ( B ) Box plots displaying sgRNA distribution in the experimental groups from lncRNA CRISPR-Cas9 library: D0-DMSO (baseline), D28-DMSO (vehicle control), and D28-Abiraterone (treatment). ( C and D ) Volcano plots showing depleted (red; RRA Score ≤ 0.05, -log□FC ≥ 2) and enriched (blue; RRA Score ≤ 0.05, log□FC ≥ 2) genes. Screening analysis was performed with MaGeCK RRA. ( C ) Negative selection identified 523 abiraterone resistance-associated LncRNAs and 553 essential LncRNAs. ( D ) Positive selection revealed 717 LncRNAs associated with abiraterone sensitivity and 169 essential LncRNAs. ( E ) Venn diagram showed negatively selected genes from two comparisons: Abiraterone vs Control and Control vs D0. ( F ) MAGeCK analysis results displayed a ranking of genes based on their RRA scores. ( G ) Frequency distribution of log2 fold change for all sgRNAs (top) and log2 fold change of individual sgRNAs for representative candidates (bottom). Enriched and depleted sgRNA hits were indicated by red and blue vertical bars, respectively. ( H ) The RRA score distribution plot revealed the top 10 candidate LncRNAs associated with abiraterone resistance. ( I-N ) Cell viability assays in PC3 ( I-K ) and DU145 ( L-N ) cells treated with 0-70 μM abiraterone for 48h, following transduction with either control sgRNAs or sgRNAs targeting candidate lncRNAs: RP11-1079K10.3 ( I and L ), WWTR1-AS1 ( J and M ), and RP11-49K24.4 ( K and N ). Data are shown as the mean ± SD (n = 4 biological replicates). Data were analyzed by two-way analysis of variance (ANOVA) with Dunnett’s multiple comparisons test ( I-N ).

Article Snippet: PC3-Cas9 cells (4×10 ) were transduced with either the Splicing-targeting CRISPR-Cas9 library for human lncRNAs (Addgene, Cat# 119977) or the Human genome-wide lentiviral CRISPR gRNA library version 1 (Addgene, Cat# 67989) at a multiplicity of infection (MOI) of 0.3, ensuring single gRNA integration per cell.

Techniques: CRISPR, Control, Selection, Transduction

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: YTHDC1 m6A-dependent and m6A-independent functions converge to preserve DNA damage response

doi: 10.1101/2024.03.25.586632

Figure Lengend Snippet: A) Schematic representation of p21 endogenously tagged reporter system in A549 cell line. B) Overview of the CRISPR-screening design. A library containing 1,828 guide RNAs was cloned into a lentiviral system and used to infect A549 reporter cells. After 4 days of infection, cells were exposed to Nutlin-3a to allow for p53 protein stabilization and subsequent reporter gene activation. Cells were sorted by FACS based on the level of reporter gene activation. C) Scatter plot of Log2 Fold Change for sgRNA enrichment in p53-enhanced-response (Y-axis) and p53-attenuated-response (X-axis) populations. The three top candidates for each population are labelled and highlighted in the plot. D) Functional annotation of the top 50 candidates for p53-enhanced and p53-attenuated populations based on their molecular activity. E) In vitro validation of the top candidate for p53-enhanced and p53-attenuated populations. Each gene was independently silenced by siRNA. After silencing, mVenus signal was taken as readout of p21-reporter gene activation. Statistical analysis was performed by paired two-tailed Student’s t-test KD versus scramble (SCR)control. F) Pearson correlation score between p53-downstream-effector misregulation (p53 pathway) compared to the expressionof the 407 CRISPR-Screening candidates in Lung carcinoma patients from TCGA (LUAD + LUSC patients) with p53 wild-type genotype versus healthy patients. Data information: All data are shown are representative of at least three independent experiments. Data are presented as mean ± s.d. ns, not significant (P ≥ 0.05), *(P ≤ 0.05), ** (P ≤ 0.01), paired two-tailed student’st-test was performed in E.

Article Snippet: We further modified p21-reporter cells to stably express SpCas9 endonuclease , and transduced these cells with human lentiviral-based CRISPR pooled library in CRISPseq-BFP-backbone vector (Addgene).

Techniques: CRISPR, Clone Assay, Infection, Activation Assay, Functional Assay, Activity Assay, In Vitro, Two Tailed Test

Journal: bioRxiv

Article Title: YTHDC1 m6A-dependent and m6A-independent functions converge to preserve DNA damage response

doi: 10.1101/2024.03.25.586632

Figure Lengend Snippet: A) Representative Immunoblot of p53, with GAPDH as loading control. Cells were treated with Nutlin-3a or untreated as negative control. B) Nutlin-3a dose response treatment. P21-Reporter cells were treated with increasing doses of Nutlin-3a or untreated as negative control. After treatment, reporter gene activation was measured by flow cytometry. The percentage of cells showing reporter gene activation was calculated and presented in the plot of the signal distribution. C) Representative immunoblot of spCas9, with a-tubulin as loading control. Cells were transduced with a lentivirus carrying spCas9 or an empty vector as negative control. D) Flow cytometry quantification of P21-Reporter gene activation and CRISPR library infection for the two replicates used in the screening. E) Scatter plot of log2 fold change for sgRNA enrichment in p53-enhanced and p53-attenuated populations. Each individual sgRNA for the three top candidates for each population is labelled and highlighted in the plot. Non-targeting sgRNAs used as negative controls in black. F) Scatter plot of log2 fold change for gene enrichment in p53-enhanced and p53-attenuated populations. Previously reported negative or positive regulators of p53 activity are labelled in the plot in red and green, respectively. Functional annotation of the top 50 candidates for p53-enhanced and p53-attenuated populations based on theirmolecular substrate. H) RNA level quantification of mature mRNA by RT-qPCR for YTHDC1 and ASH2L . Cells were transfectedwith two independent siRNA against YTHDC1 (DC1-1 and -2), two independent siRNA against ASH2L (ASH2L-1 and -2) or Scramble (SCR) as negative control. Data information: All data are shown are representative of at least three independent experiments, except for C. Data are presented as mean ± s.d. *** (P ≤ 0.001), **** (P ≤ 0.0001), paired two-tailed student’s t-testwas performed in H.

Article Snippet: We further modified p21-reporter cells to stably express SpCas9 endonuclease , and transduced these cells with human lentiviral-based CRISPR pooled library in CRISPseq-BFP-backbone vector (Addgene).

Techniques: Western Blot, Negative Control, Activation Assay, Flow Cytometry, Transduction, Plasmid Preparation, CRISPR, Infection, Activity Assay, Functional Assay, Quantitative RT-PCR, Two Tailed Test

Journal: bioRxiv

Article Title: Heterotypic inter-GPCR ß-arrestin coupling regulates lymphatic endothelial junctional architecture in murine lymph nodes

doi: 10.1101/435776

Figure Lengend Snippet: ( A ) Schematic of S1PR1 modulator screening system Four lentiviral vectors were transduced into U2OS cell line to enable gene activation by SAM and monitoring S1PR1 activation by TANGO system. The cells introduced with SAM sgRNA library were starved with 0.5% charcoal treated FBS, then the Venus-positive population was sorted and next-gen sequence (NGS) analysis was carried out to identify the enriched SAM sgRNA sequences. ( B ) Scatter plot showing enrichment of sgRNAs after sorting. Most sgRNAs are equally distributed in the pre-sort sample (closed gray circles) while after sorting a small fraction of sgRNAs (2,770 out of 70,290 sgRNAs) were enriched and others were not detected (open blue circles). The y-axis shows the NGS reads of sgRNAs. ( C ) Identification of top candidate genes using the MAGeCK method . The names of top ten candidate genes are indicated.

Article Snippet: The single clones were isolated from antibiotics resistant cells by limiting dilution, then introduced with the SAM sgRNA library (a gift from Feng Zhang, Addgene #1000000057) at a low multiplicity of infection.

Techniques: Activation Assay, Sequencing

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: 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: Science Advances

Article Title: Genome-wide CRISPR screen identifies IRF1 and TFAP4 as transcriptional regulators of Galectin-9 in T cell acute lymphoblastic leukemia

doi: 10.1126/sciadv.ads8351

Figure Lengend Snippet: ( A ) Flow cytometry analysis of Galectin-9 expression in Jurkat, CCRF-CEM, Loucy, and HPB-ALL scrambled (Scr) and Galectin-9 KO T-ALL cell lines. Representative flow plots are shown (top), and the mean fluorescence intensity (MFI) was calculated of duplicate experiments (bottom) and normalized to the average MFI of Jurkat and CCRF-CEM scrambled control cell lines. A t test was used for statistical analysis. * P < 0.05; n.s., not significant. ( B ) Schematic overview of the genome-wide CRISPR screen for Galectin-9 in T-ALL cell line Jurkat. Created in BioRender. B.Y. (2025) https://BioRender.com/n03l034 . ( C ) Scatterplot showing log 2 fold changes of 20% low versus 20% high expressing Jurkat cells in the genome-wide CRISPR screen assessed by MAGeCK. Dot size indicates −log 10 of the mean FDR of both replicates of a one-sided significance test using a negative binomial model by the MAGeCK algorithm . ( D ) Heatmap depicting the log 2 fold change of 20% low versus 20% high Galectin-9 expressing Jurkat cells in the CRISPR genome-wide and validation screen. Only significant hits of the genome-wide screen and nontargeting controls were included and shown. ( E ) Log 2 fold changes of sgRNA abundance of significant hits IRF1 , TFAP4 , and ADD1 and positive control LGALS9 in the genome-wide CRISPR screen (top) and validation CRISPR screen (bottom). ( F ) Pearson correlation of IRF1 , TFAP4 , and ADD1 expression with LGALS9 expression in the TCGA and TARGET cohorts [UCSC Xena platform ( n = 14,726) ]. See table S1 for abbreviations of cancer types and the number of patients. ( G ) Boxplots depicting log2 IRF1 , TFAP4 , and ADD1 expression (normalized DESeq2 counts+1) in primary T-ALL (TARGET cohort, n = 265) split by low, mid, and high LGALS9 expression. An unpaired t test was used for statistical analysis. ** P < 0.01; *** P < 0.001; n.s., not significant.

Article Snippet: Human lentiviral CRISPR library was a gift from X. S. Liu (Addgene no. 1000000132).

Techniques: Flow Cytometry, Expressing, Fluorescence, Control, Genome Wide, CRISPR, Biomarker Discovery, Positive Control

Journal: Science Advances

Article Title: Genome-wide CRISPR screen identifies IRF1 and TFAP4 as transcriptional regulators of Galectin-9 in T cell acute lymphoblastic leukemia

doi: 10.1126/sciadv.ads8351

Figure Lengend Snippet: ( A ) Western blot quantification in single-cell–derived CRISPR KO clones in Jurkat T-ALL cells. Intensities were normalized to GAPDH and scrambled controls. Error bars indicate SEM of duplicates for which Western blots are shown. Data of scrambled 1+2 represent both scrambled controls together. ( B ) Bar plot showing Galectin-9 concentration secreted in cell culture supernatant after 2 days of culture in Jurkat scrambled control, IRF1 KO, and TFAP4 KO cell lines. Error bars indicate SEM of triplicates. ( C ) Bar plot depicting fold change of LGALS9 expression assessed by RT-qPCR compared to scrambled control in Jurkat scrambled, IRF1 KO, and TFAP4 KO cell lines. Error bars indicate SEM of triplicates. ( D and E ) Western blot (WB) quantification (D) and fold change of LGALS9 expression assessed by RT-qPCR (E) of IRF1-TFAP4 dual KO Jurkat cells. Single-cell–derived scrambled or TFAP4 KO cell lines were subjected to CRISPR editing of IRF1 and scrambled gRNAs. A bulk IRF1 KO sample was taken along as well and note order TFAP4 KO2/1 in WB. WB intensities were normalized to GAPDH and scrambled controls. Error bars indicate SEM of duplicates (D) or triplicates (E). Data of scrambled 1+2 represent both scrambled controls together. ( F ) Bar plot depicting fold change of LGALS9 expression assessed by RT-qPCR of Jurkat cells treated with a range of IFN-γ for 48 hours. Expression was normalized to vehicle-treated scrambled control. Error bars indicate SEM of triplicates. ( G ) Heatmap depicting TF activity analysis [RScenic ] in primary pediatric T-ALL patient samples from the TARGET cohort ( n = 265). Samples were equally split in LGALS9 high, mid, and low expressing groups. See table S2 for other regulons in the same hierarchical cluster as IRF1 and TFAP4. An unpaired t test was used for all statistical analysis. * P < 0.05; ** P < 0.01; *** P < 0.001; n.s., not significant.

Article Snippet: Human lentiviral CRISPR library was a gift from X. S. Liu (Addgene no. 1000000132).

Techniques: Western Blot, Derivative Assay, CRISPR, Clone Assay, Concentration Assay, Cell Culture, Control, Expressing, Quantitative RT-PCR, Activity Assay

Journal: Science Advances

Article Title: Genome-wide CRISPR screen identifies IRF1 and TFAP4 as transcriptional regulators of Galectin-9 in T cell acute lymphoblastic leukemia

doi: 10.1126/sciadv.ads8351

Figure Lengend Snippet: ( A ) H3K27ac enrichment in the LGALS9 locus analyzed by ChIP-seq experiments in T-ALL cell lines. ( B ) Chromatin looping analyzed by H3K27ac HiChIP-seq experiments in Jurkat and CCRF-CEM cells. Only chromatin looping from the LGALS9 promoter is shown, and three enhancers detected in both cell lines are indicated by an arrow. ( C ) H3K27ac, IRF1, and TFAP4 enrichment in the LGALS9 locus analyzed by ChIP-seq experiments in Jurkat and CCRF-CEM cells. ( D ) Heatmap depicting H3K27ac (left), IRF1 (middle), and TFAP4 (right) enrichment on the LGALS9 promoter and three identified enhancers. Faint gray outline indicates that this region is not peak called in the respective sample. ( E ) Bar plot depicting fold change of LGALS9 expression assessed by RT-qPCR compared to scrambled control in single clone–derived CRISPR-edited Jurkat cells upon a 150-bp deletion in enhancer 1, a 1.6-kb deletion in enhancer 2, or scrambled controls. Error bars indicate SEM of triplicates, and an unpaired t test was used for statistical analysis; * P < 0.05; ** P < 0.01; n.s., not significant. ( F ) Schematic summary of the transcriptional regulation of LGALS9 expression. Created in BioRender. B.Y. (2025) https://BioRender.com/a56h547 .

Article Snippet: Human lentiviral CRISPR library was a gift from X. S. Liu (Addgene no. 1000000132).

Techniques: ChIP-sequencing, HiChIP, Expressing, Quantitative RT-PCR, Control, Derivative Assay, CRISPR

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