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