sgrna library system  (TaKaRa)

Journal: Scientific Reports

Article Title: Genome-wide CRISPR screen for human factors involved in alternative polyadenylation based on differential localization of CD47

doi: 10.1038/s41598-025-14782-7

Figure Lengend Snippet: Genome-wide CRISPR screening identifies candidate APA regulators . ( a ) Schematic of the genome-wide CRISPR screen based on differential CD47 localization. ( b ) FACS analysis of cell surface and intracellular CD47 expression in HeLa cells stably expressing Cas9. The 10% of cells with the lowest (CD47 surface high) and 10% with the highest (CD47 surface low) intracellular-to-cell surface CD47 expression ratios were collected. ( c ), ( d ) Gene-level enrichment of sgRNAs in CD47 surface high ( c ) and CD47 surface low ( d ) cells, with the x-axis representing log 2 fold changes and the y-axis showing MAGeCK P-values. Dashed lines indicate P < 0.05 and log 2 fold changes > 1, with significantly enriched genes highlighted. ( e ) ( f ) GO enrichment analysis of top 100 ranked genes whose depletion resulted in cell surface CD47 retention (CD47 surface high) ( e ) or intracellular CD47 accumulation (CD47 surface low) ( f ).

Article Snippet: For pooled genome-wide CRISPR screening, the Guide-it CRISPR Genome-Wide sgRNA Library (Takara), comprising 76,612 sgRNAs targeting 19,114 human genes, was packaged into lentivirus following the manufacturer’s protocol.

Techniques: Genome Wide, CRISPR, Expressing, Stable Transfection

Journal: Scientific Reports

Article Title: Genome-wide CRISPR screen for human factors involved in alternative polyadenylation based on differential localization of CD47

doi: 10.1038/s41598-025-14782-7

Figure Lengend Snippet: Secondary screen identifies candidate genes potentially involved in APA regulation. ( a ) Schematic of the secondary CRISPR screen based on differential CD47 localization. ( b ) Quantification of MFI values from FACS analysis of CD47. The double staining approach was applied to HeLa cells stably expressing control shRNA or shRNA targeting candidate genes. Results are presented as mean ± s.d. ****, P < 0.0001; ***, P < 0.001; **, P < 0.01 (two-tailed t-test for independent samples, n = 3). ( c ) qRT-PCR analysis of total CD47 mRNA and the long 3′ UTR isoform in HeLa cells stably expressing control shRNA or shRNA targeting candidate genes. Results are presented as mean ± s.d. ****, P < 0.0001; ***, P < 0.001; **, P < 0.01 (two-tailed t-test for independent samples, n = 3).

Article Snippet: For pooled genome-wide CRISPR screening, the Guide-it CRISPR Genome-Wide sgRNA Library (Takara), comprising 76,612 sgRNAs targeting 19,114 human genes, was packaged into lentivirus following the manufacturer’s protocol.

Techniques: CRISPR, Double Staining, Stable Transfection, Expressing, Control, shRNA, Two Tailed Test, Quantitative RT-PCR

Journal: Scientific Reports

Article Title: Genome-wide CRISPR screen for human factors involved in alternative polyadenylation based on differential localization of CD47

doi: 10.1038/s41598-025-14782-7

Figure Lengend Snippet: Genome-wide CRISPR screening identifies candidate APA regulators . ( a ) Schematic of the genome-wide CRISPR screen based on differential CD47 localization. ( b ) FACS analysis of cell surface and intracellular CD47 expression in HeLa cells stably expressing Cas9. The 10% of cells with the lowest (CD47 surface high) and 10% with the highest (CD47 surface low) intracellular-to-cell surface CD47 expression ratios were collected. ( c ), ( d ) Gene-level enrichment of sgRNAs in CD47 surface high ( c ) and CD47 surface low ( d ) cells, with the x-axis representing log 2 fold changes and the y-axis showing MAGeCK P-values. Dashed lines indicate P < 0.05 and log 2 fold changes > 1, with significantly enriched genes highlighted. ( e ) ( f ) GO enrichment analysis of top 100 ranked genes whose depletion resulted in cell surface CD47 retention (CD47 surface high) ( e ) or intracellular CD47 accumulation (CD47 surface low) ( f ).

Article Snippet: For pooled genome-wide CRISPR screening, the Guide-it CRISPR Genome-Wide sgRNA Library (Takara), comprising 76,612 sgRNAs targeting 19,114 human genes, was packaged into lentivirus following the manufacturer’s protocol.

Techniques: Genome Wide, CRISPR, Expressing, Stable Transfection

Journal: Scientific Reports

Article Title: Genome-wide CRISPR screen for human factors involved in alternative polyadenylation based on differential localization of CD47

doi: 10.1038/s41598-025-14782-7

Figure Lengend Snippet: Secondary screen identifies candidate genes potentially involved in APA regulation. ( a ) Schematic of the secondary CRISPR screen based on differential CD47 localization. ( b ) Quantification of MFI values from FACS analysis of CD47. The double staining approach was applied to HeLa cells stably expressing control shRNA or shRNA targeting candidate genes. Results are presented as mean ± s.d. ****, P < 0.0001; ***, P < 0.001; **, P < 0.01 (two-tailed t-test for independent samples, n = 3). ( c ) qRT-PCR analysis of total CD47 mRNA and the long 3′ UTR isoform in HeLa cells stably expressing control shRNA or shRNA targeting candidate genes. Results are presented as mean ± s.d. ****, P < 0.0001; ***, P < 0.001; **, P < 0.01 (two-tailed t-test for independent samples, n = 3).

Article Snippet: For pooled genome-wide CRISPR screening, the Guide-it CRISPR Genome-Wide sgRNA Library (Takara), comprising 76,612 sgRNAs targeting 19,114 human genes, was packaged into lentivirus following the manufacturer’s protocol.

Techniques: CRISPR, Double Staining, Stable Transfection, Expressing, Control, shRNA, Two Tailed Test, Quantitative RT-PCR

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