Journal: Science advances

Article Title: EIF3D safeguards the homeostasis of key signaling pathways in human primed pluripotency.

doi: 10.1126/sciadv.adq5484

Figure Lengend Snippet: Fig. 1. EIF3D is essential for maintaining primed pluripotency. (A) Rank plot from CRISPRi screening. Red and blue dots represent genes significantly increased or decreased (±1 SD) following 8 and 16 days of KD, respectively. n = 3. The full list is provided in data S1 and S2. (B) Protein expression in undifferentiated and differentiating PSCs (10 and 20 days post-FGF withdrawal) and HDFs. Vinculin (VCL) was used as a loading control. (C) Representative images of control and EIF3D KD iPSCs, 5 days post- KD induction. Scale bars, 100 μm. (D) Cell counts on days 3 (P = 4.70 × 10−4) and 5 (P = 3.53 × 10−7) post-KD induction (mean ± SD, n = 6). P values determined via unpaired t test. (E) Cell cycle phase distribution (mean ± SD, n = 3). G1: P = 2.14 × 10−3; S: P = 4.43 × 10−7; G2/M: P = 1.99 × 10−4, calculated by unpaired t test. (F) RNA expression of pluripotency and p53-related genes during EIF3D KD. n = 3. (G) Expression of pluripotency and p53-associated proteins during EIF3D KD. d, days; FC, fold change.

Article Snippet: Genome- wide CRISPRi screens Ten micrograms of the genome- wide CRISPRi library hCRISPRi- v2 (courtesy of J. Weissman; Addgene, #83969) which contains five sgRNA for each of 18,905 mRNAs along with 3.75 μg of psPAX2 (courtesy of D. Trono; Addgene, #12260) and 1.25 μg of pMD2.G (courtesy of D. Trono; Addgene, #12259) were transfected into 293T/17 cells (passage 27) using TransIT- Lenti Transfection Reagent (Mirus).

Techniques: Expressing, Control, RNA Expression

Journal: International Journal of Molecular Sciences

Article Title: Genome-Wide CRISPR Screens Identify ABCG2-Mediated Drug Resistance to the Threonine Tyrosine Kinase (TTK) Inhibitor CFI-402257 in Breast Cancer

doi: 10.3390/ijms27062665

Figure Lengend Snippet: CRISPR activation screens identify ABCG2 as a putative 2257 resistance-conferring gene in TNBC cell lines. ( A ) Schematic indicating the design of genome-wide CRISPRa screens to discover genes whose GOF confers resistance to 2257 in TNBC models. sgRNAs induce expression of target genes in dCas9-VPR+ cells. ( B , C ) Visualization of screen hits identified using the Drug Z algorithm in MDA-MB-231 ( B ) and MDA-MB-436 ( C ) cells treated with an IC 95 dose of 2257. Rank indicates the rank of individual genes assessed in the screen, and normalized Z-score represents a composite score indicating the relative abundance of all sgRNAs targeting specific genes in 2257-treated cells compared to cells collected at the time of screen onset. Positive Z-scores indicate sgRNAs that are enriched in 2257-treated cells. ABCG2 was the top hit identified in both cell lines. ( D , E ) sgRNAs targeting ABCG2 comprise a substantial fraction of total sgRNA sequencing reads in the MDA-MB-231 and MDA-MB-436 CRISPRa screens. The percentage of total sgRNA reads obtained for each 2257-treated replicate at the end of the screen is plotted for each sgRNA targeting ABCG2 in the hCRISPRa-v2 sgRNA library. sgRNA abundances at T0 are not shown because they were <0.1% of reads in both cell lines.

Article Snippet: The Weissman genome-wide CRISPR activation (CRISPRa) library containing 104,540 sgRNAs targeting 19,915 human genes was obtained from Addgene (hCRISPRa-v2; #83978) [ ].

Techniques: CRISPR, Activation Assay, Genome Wide, Expressing, Sequencing

Journal: Nature

Article Title: Lysosome-targeting chimaeras for degradation of extracellular proteins

doi: 10.1038/s41586-020-2545-9

Figure Lengend Snippet: a, Schematic of a CRISPRi screen in K562 cells stably expressing dCas9-KRAB and a library of sgRNAs with genome-wide coverage. b, Selected gene hits for regulation of NA-647 internalization by LYTACs. c, Gene ontology (GO) annotation for significant hits (<5% FDR). d, e, Cell surface expression levels of CI-M6PR in dCas9-KRAB K562 cells (d) or dCas9-KRAB HeLa cells (e) transfected with control sgRNA, IGF2R-targeting sgRNA, EXOC1-targeting sgRNA or EXOC2-targeting sgRNA. Cells were stained for CI-M6PR and subjected to live-cell flow cytometry. f, g, Western blot analysis of EXOC1 and CI-M6PR in K562 (f) and HeLa (g) CRISPRi knockdown lines. For c, analysis is representative of two replicates; d, f, g are representative of two independent experiments. K562 cells rapidly lost the exocyst knockdown phenotype after two passages. For e, data are mean ± s.d. of three independent experiments in HeLa and normalized to non-targeting sgRNA. P values were determined by unpaired two-tailed t-tests.

Article Snippet: CRISPRi screen procedure The hCRISPRi-v2 library was a gift from J. Weissman (Addgene ID 83969).

Techniques: Stable Transfection, Expressing, Genome Wide, Transfection, Control, Staining, Flow Cytometry, Western Blot, Knockdown, Two Tailed Test

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) Cells were untreated or pre-treated with the GRK2/3 inhibitor Compound 101 (10 or 20 μM) for 30 min and then stimulated with either 100 nM or 1 μM fMLF for 10 min (n=6). (B) dCas9 expressing dHL-60s (Control) and Compound 101 pretreated dHL-60 cells expressing dCas9 and GRK6 sgRNA#1 were stimulated with AF488-fMLF for 5 min, fixed, and stained with an anti-FPR1 antibody (scale bar, 5μm). Control and CRISPRi knockdown cells were untreated or pre-treated with Compound 101 for 30 min and then stimulated with either (C) 100 nM or (D) 1 μM fMLF for 10 min (n=6). ‘g’ stands for sgRNA, and 1 and 2 are unique guide sequences. (E) Primary human neutrophils were untreated or pre-treated with 20 μM Compound 101 for 30 min and then stimulated with 100 nM fMLF for 10 min (n=4). Throughout this study, we use Mann-Whitney U test for pairwise comparison of the mean fluorescence signal (bar height) of the two samples indicated in figures (n.s. p>0.05, *p<0.05, **p<0.01, ***p<0.001), and dots represent different biological replicates. (F) Schematic summary of how defects in different GRK activities affect FPR1 internalization.

Article Snippet: Single (g1 or g2) CRISPRi knock-downs of GRK2, GRK3, GRK5, and GRK6 were made by stable co-expression of a dCas9 construct (SID4x- dCas9-XTEN80-KRAB) and the guides (ranked #1 and #2 at Weissman Lab hCRISPRi-v2 library, Addgene #1000000090, Supplementary Table 8) through lentiviral infection.

Techniques: Expressing, Control, Staining, Knockdown, MANN-WHITNEY, Comparison, Fluorescence