Journal: Molecular cell

Article Title: ZC3H14 facilitates backsplicing by binding to exon-intron boundary and 3' UTR.

doi: 10.1016/j.molcel.2024.10.001

Figure Lengend Snippet: Figure 1. Genome-wide CRISPR screen for regulators of circRNA biogenesis with circReporter cells (A) Illustration of circReporter cell construction. The circGFP minigene included circVN1R1 flanking sequences, inverted split-GFP fragments, and IRES. (B) GFP and mCherry protein levels in circReporter cells treated with siRNA (sicircGFP) against circGFP backsplicing junction (BSJ). siNC, negative control siRNA with scrambled sequences. (C) Procedure of genome-wide CRISPR knockout screen. (D) Overlapped positive and negative regulators identified from GFPhigh and GFPlow cells. (E) GO analysis of 83 overlapped regulators identified from GFPhigh and GFPlow cells. (F) 20 regulators enriched in the RNA-binding GO term.

Article Snippet: Genome-wide CRISPR knockout screen and data analysis The Human CRISPR knockout pooled library (Brunello) was purchased from the Addgene (https://www.addgene.org/).

Techniques: Genome Wide, CRISPR, Negative Control, Knock-Out, RNA Binding Assay

Journal: Genome Research

Article Title: Genome-scale identification of cellular pathways required for cell surface recognition

doi: 10.1101/gr.231183.117

Figure Lengend Snippet: A cell-based genome-scale CRISPR-KO approach identifies pathways required for monoclonal antibody surface epitope recognition. ( A ) Schematic of the approach based on CRISPR/Cas9 technology using a genome-scale lentiviral gRNA library. Genes identified as being required for surface display of the epitope recognized by an anti-CD58 ( B ) and anti-GYPA ( C ) mAbs. The enrichment of gRNAs targeting each gene is quantified as the robust rank aggregation (RRA) score calculated using the MAGeCK software between selected cells that had lost the mAb epitope versus control cells and is shown plotted in rank order. ( D ) Genes identified as being required for surface display of the epitope recognized by an anti-CD59 mAb; here, genes are ordered alphabetically for clarity. Circles represent individual genes and are sized according to their false-discovery rate (FDR): large circle = FDR < 1%, small circle = 1% < FDR < 5%. Genes encoding the direct receptors are indicated with gray circles. Only genes with FDR < 5% are named and are color-coded according to their function.

Article Snippet: The Human Improved Genome-Wide Knockout CRISPR Library v1 (Addgene no. 67989), lentiviral Cas9 reporter plasmids pKLV2-U6gRNA5(gGFP)-PGKBFP2AGFP-W (Addgene no. 67980) and pKLV2-U6gRNA5(Empty)-PGKBFP2AGFP-W (Addgene no. 67979), lentiviral vector expressing Cas9 fused with the blasticidin-resistant gene at the C terminus pKLV2-EF1a-Cas9Bsd-W (Addgene no. 68343), and lentiviral CRISPR gRNA expression vector pKLV2-U6gRNA5(BbsI)-PGKpuro2ABFP-W (Addgene no. 67974) were used in this study.

Techniques: CRISPR, Software, Control

Journal: bioRxiv

Article Title: CRISPR activation screens map the genomic landscape of cancer glycome remodeling

doi: 10.1101/2025.05.26.656133

Figure Lengend Snippet: A) Inhibition of anticancer immunity by Siglec receptors. Siglecs bind to sialic acid-containing glycans on the surface of target cells. B) Components of the Siglec-7 ligand structure. Disialyl-T glycans are organized in dense arrays on the backbone of specific mucin-type O-glycoproteins. C ) Targeted overexpression of genes by CRISPRa. A cell line expressing a dCas9-GCN4 peptide array and a VP64-SunTag fusion protein serves as the target cell line (K-562-CRISPRa cells). This cell line is lentivirally transduced with an sgRNA targeting the promoter region of a target gene. Subsequent recruitment of chromatin remodeling factors induces an increase in transcriptional activity. D) Recombinant Siglec-Fc proteins were precomplexed with an AlexaFluor647-antihuFc antibody (1 μg/mL) for 1 hour on ice. Precomplexes were subsequently incubated with K-562 cells for 30 minutes and analyzed by flow cytometry. Representative flow cytometry plots are depicted for each Siglec-Fc as well as a human Fc (hFc) negative control. E) K-562-CRISPRa cells were lentivirally transduced with a genome-wide library of ∼104,000 sgRNAs (5 sgRNAs/gene). After selection and propagation, 1.25 x 10 8 cells were then stained with Siglec-Fc reagents as in B . A high-binding (top 20%) and low-binding (bottom 20%) of the fluorescent population was then selected and sorted by FACS.

Article Snippet: The CRISPRa-v2 library (top 5 sgRNAs/gene) containing 104,535 sgRNAs was purchased from Addgene (Pooled Library 83978).

Techniques: Inhibition, Over Expression, Expressing, Peptide Microarray, Transduction, Activity Assay, Recombinant, Incubation, Flow Cytometry, Negative Control, Genome Wide, Selection, Staining, Binding Assay

Journal: bioRxiv

Article Title: CRISPR activation screens map the genomic landscape of cancer glycome remodeling

doi: 10.1101/2025.05.26.656133

Figure Lengend Snippet: A) K-562-dCas9-CRISPRa cells were transduced with two different sgRNAs targeting the CD24 promoter region. After selection with puromycin, cells were stained with Siglec-10-Fc as in 1B and with a fluorescent antibody against CD24. Representative flow cytometry plots for both stains are shown. B) THP-1 cells were stained with Siglec-10-Fc as in 1B and a fluorescent antibody against CD24. Representative flow cytometry plots for both stains are shown. C) OCI-AML-2-Cas9 cells were lentivirally transduced with an sgRNA targeting MGAT1 to generate a polyclonal cell population containing WT and MGAT1 KO cells. Cells were then co-stained with Siglec-10-Fc and the lectin L-PHA (5 μg/mL), which binds to complex N-linked glycans. Representative staining is indicated on the flow cytometry plot. D) Graph indicates MFI of Siglec-10-Fc staining in the WT (L-PHA+) and MGAT1 KO (L-PHA-) populations. E) OCI-AML-2-Cas9 cells were lentivirally transduced with an sgRNA targeting C1GALT1. Cells were co-stained with Siglec-10-Fc and the lectin DBA, which binds to exposed α-GalNAc. Representative staining is indicated on the flow cytometry plot. F) Graph indicates MFI of Siglec-10-Fc staining in the WT (DBA-) and C1GALT1 KO (DBA+) populations. G) Pathway diagram indicates the enzymes involved in 2,3-linked and 2,6-linked sialylation of N-linked glycans. ST3GAL4, ST3GAL6 and ST6GAL1 were all strong hits in the Siglec-10 screen. H) The heat map indicates the average mRNA expression of key glycosyltransferase hits in cell lines derived from B-ALL, T-ALL, AML and multiple myeloma. mRNA expression values were extracted from DepMap and the Human Protein Atlas. I) OCI-AML-2-Cas9 cells were transduced an sgRNAs against ST3GAL4. Cells were then stained with fluorescently labeled Siglec-10-Fc. A representative flow cytometry plot is shown. J) The MFI of Siglec-10 staining in OCI-AML-2 WT and ST3GAL4 KO cells is indicated. MFI is internally normalized to the WT cell line. K) MM1S-Cas9 cells were transduced an sgRNAs against ST6GAL1. ST6GAL1 KO cells were isolated by FACS using the lectin SNA. Cells were then stained with fluorescently labeled Siglec-10-Fc. A representative flow cytometry plot is shown. L) The MFI of Siglec-10 staining in MM1S WT and ST6GAL1 KO cells is indicated. MFI is internally normalized to the WT cell line. M) K-562-CRISPRa cells were transduced and stained as in 4C . A representative flow cytometry plot is shown comparing Siglec-10-Fc staining in cells transduced with a non-targeting (NT) sgRNA to cells within the “CD44 High” expression gate. N) The MFI of Siglec-10-Fc staining in NT cells vs. “CD44 High” cells is shown. MFI is internally normalized to the WT cell line. O) K-562 CRISPRa cells were transduced with an sgRNA against CSPG4 and co-stained with Sig10-Fc and a CSPG4 antibody as in 4C . A representative flow cytometry plot is shown comparing Siglec-10-Fc staining in cells transduced with a non-targeting (NT) sgRNA to cells within the “CSPG4 High” expression gate. P) The MFI of Siglec-10 staining in NT cells vs. “CSPG4 High” cells is shown. MFI is internally normalized to the WT cell line. Statistical significance was determined using a two-tailed t-test. ** indicates p<0.01, * indicates p<0.05. Mean values plotted, error bars indicate SEM.

Article Snippet: The CRISPRa-v2 library (top 5 sgRNAs/gene) containing 104,535 sgRNAs was purchased from Addgene (Pooled Library 83978).

Techniques: Transduction, Selection, Staining, Flow Cytometry, Expressing, Derivative Assay, Labeling, Isolation, Two Tailed Test

Journal:

Article Title: NOEY2 (ARHI), an imprinted putative tumor suppressor gene in ovarian and breast carcinomas

doi:

Figure Lengend Snippet: Metaphase mapping of NOEY2 by fluorescence in situ hybridization. A P1 clone containing NOEY2 was mapped to 4′,6-diamidino-2-phenylindole-banded human chromosome 1p31 in normal human lymphocytes. (Inset) Localization of NOEY2 (green) on chromosome 1 counterstained with propidium iodide and 4′,6-diamidino-2-phenylindole compared with 4′,6-diamidino-2-phenylindole-banded chromosome 1.

Article Snippet: The NOEY2 gene was mapped to chromosome 1p31 by screening a P1 genomic library (DuPont) with two sets of primers that encompass the entire coding and 3′ untranslated region.

Techniques: Fluorescence, In Situ Hybridization