genome wide crispr knockout pooled library screen human geckov2 crispr knockout pooled libraries  (Addgene inc)

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: PLOS One

Article Title: Conformation of HLA-E/peptide complex guides interaction with two novel HLA-E receptors: Stabilin 1 and 2

doi: 10.1371/journal.pone.0334543

Figure Lengend Snippet: (A) Schematic overview of the receptor-ligand CRISPR activation screen. K562 cells stably expressing dCas9 were transduced with a genome-wide activation library and stained with a pool of HLA-E*01:03 tetramers loaded with 4 different peptides (VLRPGGHFL, RMPPLGHEL, VMAPRTLIL, RLPAKAPLL). Enrichment of gRNAs in sorted cell populations were determined using next generation sequencing. (B) Gene ranking scores of genes in two replicate screens were calculated using SigmaFC scores from PinAPL-Py and plotted against each other. Hits of interest are annotated. (C) Enrichment of single gRNAs for top hits of the screen, red and blue stripes represent enrichment of individual gRNAs of two replicate sorts compared to unsorted cells. (D) K562 dCas9 cells were transduced with a control guide or a gRNA upregulating either STAB1 or STAB2, stained with HLA-A, -B, -C or -E tetramers (HLA-A*02:01 NLVPMVATV, HLA-B*07:02 TPRVTGGGAM, HLA-C*07:01-VRIGHLYIL, HLA-E*01:03 RMPPLGHEL) and analysed by flow cytometry. (E) K562 gSTAB1 or K562 gSTAB2 cells were stained with HLA tetramers (HLA-A*02:01; HLA-B*07:02; HLA-E*01:03) loaded with indicated peptides and analysed by flow cytometry. All graphs represent at least two biological replicates.

Article Snippet: CRISPR activation screens were performed by transducing K562 cells with dCas9-Blast (Addgene #61425), a kind gift from Feng Zhang, and the Calabrese genome-wide activation library (sublibrary A + B), kindly provided by John Doench (Addgene # 1000000111).

Techniques: CRISPR, Activation Assay, Stable Transfection, Expressing, Transduction, Genome Wide, Staining, Next-Generation Sequencing, Control, Flow Cytometry