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

Journal: Nature Communications

Article Title: A genome-wide CRISPR screen in Anopheles mosquito cells identifies fitness and immune cell function-related genes

doi: 10.1038/s41467-025-65304-y

Figure Lengend Snippet: a Schematic for the fitness gene CRISPR screen. CRISPR GuideXpress was used to design a whole-genome sgRNA library targeting protein-coding and non-coding Anopheles gambiae genes. The library was cloned into the pLib6.4B_695 vector and delivered to Sua-5B-IE8-Act::Cas9-2A-Neo via ΦC31 recombination-mediated cassette exchange to yield a pool of knockout cells. During outgrowth, cells that received sgRNAs targeting fitness genes will “drop out” of the KO pool. The relative abundance of each sgRNA in the KO outgrowth pool of cells was compared to the plasmid library by NGS followed by MAGeCK MLE analysis. b Genome-wide library coverage. The starting library design includes 90,208 sgRNAs (88,763 unique sgRNAs) targeting 93% of Anopheles genes, with 7 sgRNAs per gene coverage for ~96% of these genes. Following the screen assay, data were analyzed using MAGeCK MLE. Shown in ( c ) is the cumulative distribution of false discovery of fitness genes (genes with low expression, TPM < 1) for each replicate. Replicate 1 identified a larger number of fitness genes at a low false discovery cut-off (1280 genes at FDR = 0.05). The distribution of genes by category is shown in ( e ). All data points within the Z-score whole genome distribution for replicate 1 are displayed (gray or colored circles); colors highlight genes annotated with the indicated Gene Ontology term (Cytoplasmic Ribosome KEGG:aga030008; Mitochondrial Ribosome GO:0098798,0005763; Spliceosome KEGG:aga03040; Proteasome KEGG:aga03050). The red line intercept of the x axis represents Z-score threshold at FDR = 0.05. f Comparison of fitness genes identified in Anopheles and Drosophila . To compare gene lists between the two species, Anopheles genes were mapped to corresponding Drosophila orthologs, then scores from Anopheles replicate 1 or Drosophila knockout screens were plotted. Colored box within the plot highlights Anopheles fitness genes with Z-scores within FDR = 0.05 in replicate 1; fitted linear trendline and R 2 squared value are displayed to highlight the correlation trend between datasets; yps datapoint was darkened to enhance its visibility. Image in a was created using BioRender. Mameli, E. (2025) https://BioRender.com/rkhue0x . Source data are provided as a Source Data file.

Article Snippet: Plasmids and plasmid libraries associated with the CRISPR screens are available on Addgene (#176668 and #234477). are provided with this paper.

Techniques: CRISPR, Clone Assay, Plasmid Preparation, Knock-Out, Genome Wide, Expressing, Comparison

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