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

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: bioRxiv

Article Title: High-throughput Genome Wide CRISPR Knock Out mechanical sort identifies genes driving metastatic cancer cell softening

doi: 10.64898/2026.02.12.705447

Figure Lengend Snippet: Cancer cells expressing Cas9 are transduced with a lentiviral library containing sgRNAs across the entire protein-coding genome at a low MOI to allow for single gene knockouts. Antibiotic selection ensures only cells with gene knockouts are included in mechanical screen. Next, the library of cells with single gene knockouts are processed with a microfluidic stiffness-based cell sorting device into 5 mechanical subsets. The inset shows a side and top view of the ridged region of the device where cells must deform underneath each diagonal ridge. Softer cells deform easily underneath the ridges and follow hydrodynamic streamlines to provide a slight negative deflection towards outlets 1 and 2. Stiffer cells resist deformation and are deflected along the diagonal ridge resulting in trajectories towards outlets 4 and 5. Genomic DNA is harvested from each mechanical subset, the sgRNA regions are amplified and sequenced, and the distribution of sgRNAs before and after the mechanical screen is used to determine the correlation of gene knockouts with mechanical subsets.

Article Snippet: The human CRISPR Brunello lentiviral pooled library was designed to optimize on-target activity and reduce off-target effects (Addgene 73178-LV) .

Techniques: Expressing, Transduction, Selection, FACS, Amplification

Journal: mBio

Article Title: Evolutionary dynamics of heparan sulfate utilization by SARS-CoV-2

doi: 10.1128/mbio.01303-25

Figure Lengend Snippet: ACE2-independent binding of the Omicron RBD to cell surface HS. ( A ) CRISPR KO library screening scheme to identify Omicron RBD binding molecules expressed on HEK293T cells. ( B ) Binding of CRISPR KO library-transduced HEK293T cells with (red line) or without (shaded gray) Omicron RBD-Fc, before and after sorting. ( C ) Number of sgRNA sequences identified in Omicron RBD-Fc non-binding cells after sorting. Red: GAG-synthesis-related genes; black: other expressed genes; gray: non-expressed genes in HEK293T cells. ( D ) HS biosynthetic pathway highlighting SLC35B2 and B3GAT3. PAPS, 3'-phosphoadenosine-5'-phosphosulfate; Xyl, xylose; Gal, galactose; GlcNAc, N-acetylglucosamine; GlcA, glucuronic acid; IdoA, iduronic acid. ( E ) Binding of BA.1 RBD-Fc, anti-HS Ab, or anti-CS Ab to mock (black line), or SLC35B2 or B3GAT3 KO (red line) HEK293T cells. ( F ) Binding of WT or BA.1 RBD-Fc, PILRα-Fc, anti-HS Ab, or anti-CS Ab to ACE2 KO HEK293T cells pretreated with (+) or without (–) heparinase or chondroitinase. ( G ) Binding of WT or BA.1 RBD-Fc to HEK293T cells at different heparin concentrations. RBD-Fc binding was normalized to binding in the absence of heparin. ( H ) Immunofluorescence of human nasal tissue with anti-HS Ab and 4', 6-diamidino-2-phenylindole (DAPI). Scale bar, 200 µm. ( I ) Binding of WT or BA.1 RBD-Fc, anti-ACE2 Ab, or anti-HS Ab to cell lines. Data are mean ± SEM of three to four technical replicates. Statistical analysis was performed using two-way ANOVA with Sidak’s multiple comparison tests in panel F ; * P < 0.05, ** P < 0.01, and **** P < 0.0001; ns, not significant. Data are representative of two to three independent experiments.

Article Snippet: CRISPR KO library-transduced HEK293T cells were generated by lentivirus-mediated transduction using the Human CRISPR KO Pooled Library (GeCKO v2; Addgene, 1000000048).

Techniques: Binding Assay, CRISPR, Library Screening, Immunofluorescence, Comparison