crispra screening  (Addgene inc)

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

Journal: iScience

Article Title: CRISPR-Cas9 screening reveals a distinct class of MHC-I binders with precise HLA-peptide recognition

doi: 10.1016/j.isci.2024.110120

Figure Lengend Snippet: Receptor-ligand CRISPR-Cas9 activation screen reveals that CD55 interacts with HLA-C∗07:01-VRIG tetramers (A) Schematic of the receptor ligand CRISPR-Cas9 activation screen. K562 cells transduced with a genome-wide activation library were stained with a pool of three HLA tetramers (HLA-A∗02:01-NLVP, HLA-B∗07:02-TPRV, and HLA-C∗07:01-VRIG), and enriched gRNAs in stained cells were identified using NGS. (B) SigmaFC scores of genes from two replicate screens. SigmaFC scores were calculated using PinAplPy, and top hits are annotated. (C) K562 cells stably expressing dCas9 and transduced with a gRNA upregulating CD55 or a control guide were stained with the HLA-A, -B, -C, or tetramers as in (A) or with HLA-E∗01:01-VMAP tetramers and analyzed by flow cytometry. (D) In vitro co-immunoprecipitation of recombinant CD55-Fc with HLA-A∗02:01-NLVP, HLA-B∗07:02-TPRV, HLA-C∗07:01-VRIG, or HLA-E∗01:01-VMAP tetramers. (E) Three different cell lines (HeLa, PC-3M, or SiHa) that express CD55 endogenously were stained for CD55 (top) or with HLA-C∗07:01-VRIG tetramers (bottom) and analyzed by flow cytometry. (F) HeLa wild-type or HeLa CD55 KO cells were stained with αCD55 or HLA-C∗07:01-VRIG tetramers and analyzed by flow cytometry. All data except (B) represent at least three independent experiments. CRISPRa, CRISPR activation screen; TMs, tetramers; WT, wild-type; KO, knockout. Related to Figure S1 and Table S1 .

Article Snippet: For CRISPR activation screening for transmembrane interactors of HLA, K562 cells were transduced 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, Transduction, Genome Wide, Staining, Stable Transfection, Expressing, Control, Flow Cytometry, In Vitro, Immunoprecipitation, Recombinant, Knock-Out

Journal: iScience

Article Title: CRISPR-Cas9 screening reveals a distinct class of MHC-I binders with precise HLA-peptide recognition

doi: 10.1016/j.isci.2024.110120

Figure Lengend Snippet: CRISPR-Cas9 activation and KO screens identify an interaction between HLA-C∗07:02-YRFR and heparan sulfate chains (A) HeLa cells were stained with a variety of HLA-C∗07:02 tetramers loaded with different peptides and analyzed by flow cytometry. (B) The indicated cell lines were stained with HLA-C∗07:02-YRFR tetramers and analyzed by flow cytometry. Staining is normalized to unstained levels of that cell line. (C) Schematic of CRISPR-Cas9 KO screen in MelJuSo cells. Cells were stained with HLA-C∗07:02-YRFR, and enriched gRNAs in non-binding cells were identified using NGS. (D) SigmaFC scores of genes from two replicate screens. SigmaFC scores were calculated using PinAplPy, and genes involved in the heparan sulfate biosynthesis pathway are depicted in purple. Hits used for further validation are annotated. (E) Schematic of the CRISPR-Cas9 activation screen. K562 cells stably expressing dCas9 transduced with a genome-wide activation library were stained with HLA-C YRFR∗07:02 tetramers, and enriched gRNAs in positive cells were identified. (F) SigmaFC scores of genes from two replicate screens. SigmaFC scores were calculated using PinAplPy, and top hits are annotated. Proteoglycans of interest are depicted in purple. CRISPRa, Crispr activation screen. Related to Figure S3 , Table S1 and .

Article Snippet: For CRISPR activation screening for transmembrane interactors of HLA, K562 cells were transduced 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, Staining, Flow Cytometry, Binding Assay, Biomarker Discovery, Stable Transfection, Expressing, Transduction, Genome Wide

Journal: iScience

Article Title: CRISPR-Cas9 screening reveals a distinct class of MHC-I binders with precise HLA-peptide recognition

doi: 10.1016/j.isci.2024.110120

Figure Lengend Snippet:

Article Snippet: For CRISPR activation screening for transmembrane interactors of HLA, K562 cells were transduced 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: Virus, Recombinant, Blocking Assay, Genome Wide, Activation Assay, CRISPR, Knock-Out, Mutagenesis, Plasmid Preparation, Software, Imaging