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: 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: 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