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: Antibodies used in this study were anti-heparan sulfate chains (AMSBIO, F58-10E4), FITC anti-human CD55 (Biolegend, 311306), anti-human CD55 BRIC110 (ARP, 08-9402-2, targets SCR2 of CD55), anti-human CD55 BRIC216 (Biorad, MCA914T, targets SCR3 of CD55), anti-human CD55 MAB2009 (R&D systems, MAB2009-SP, targets SCR1 of CD55), anti-SDC2 APC (R&D systems, FAB2965A), anti-SDC4 APC (R&D systems, FAB29181A), anti-CD55 APC (Biolegend, #311311), or goat-anti-mouse APC (Biolegend, #405308).

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: Interaction of CD55 with HLA-C∗07:01-VRIG tetramers is allotype and peptide specific (A) HEK293T cells were transfected with a plasmid containing GFP and a truncation mutant of CD55 and analyzed by flow cytometry. GFP+ positive cells were analyzed for staining with HLA-C∗07:01-VRIG. Each mutant removes an additional SCR domain from CD55. Data are represented as mean ± SD. (B) HeLa cells were stained with HLA-C∗07:01-VRIG tetramers after pre-incubation with CD55 blocking antibodies targeting different SCR domains on CD55 and analyzed by flow cytometry. (C) HeLa cells were stained with either HLA-C∗07:01 or HLA-C∗07:02 tetramers loaded with the VRIG peptide and analyzed by flow cytometry. (D) HeLa cells were stained with HLA-C∗07:01 tetramers loaded with different alanine mutants of the VRIGHLYIL peptide and analyzed by flow cytometry. (E) CD55-Fc was immobilized on a Prot-G chip for SPR data using HLA-C∗07:01-VRIG tetramers as analyte to determine interaction on and off rates and K D . Response units were measured with increasing concentrations of HLA-C∗07:01-VRIG tetramers. All data represent at least three independent experiments, except (E), which represents a biological duplicate. FL, full length. Related to Figure S2 , Tables S2 and .

Article Snippet: Antibodies used in this study were anti-heparan sulfate chains (AMSBIO, F58-10E4), FITC anti-human CD55 (Biolegend, 311306), anti-human CD55 BRIC110 (ARP, 08-9402-2, targets SCR2 of CD55), anti-human CD55 BRIC216 (Biorad, MCA914T, targets SCR3 of CD55), anti-human CD55 MAB2009 (R&D systems, MAB2009-SP, targets SCR1 of CD55), anti-SDC2 APC (R&D systems, FAB2965A), anti-SDC4 APC (R&D systems, FAB29181A), anti-CD55 APC (Biolegend, #311311), or goat-anti-mouse APC (Biolegend, #405308).

Techniques: Transfection, Plasmid Preparation, Mutagenesis, Flow Cytometry, Staining, Incubation, Blocking Assay

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: Antibodies used in this study were anti-heparan sulfate chains (AMSBIO, F58-10E4), FITC anti-human CD55 (Biolegend, 311306), anti-human CD55 BRIC110 (ARP, 08-9402-2, targets SCR2 of CD55), anti-human CD55 BRIC216 (Biorad, MCA914T, targets SCR3 of CD55), anti-human CD55 MAB2009 (R&D systems, MAB2009-SP, targets SCR1 of CD55), anti-SDC2 APC (R&D systems, FAB2965A), anti-SDC4 APC (R&D systems, FAB29181A), anti-CD55 APC (Biolegend, #311311), or goat-anti-mouse APC (Biolegend, #405308).

Techniques: Virus, Recombinant, Blocking Assay, Genome Wide, Activation Assay, CRISPR, Knock-Out, Mutagenesis, Plasmid Preparation, Software, Imaging

Journal: The Journal of Cell Biology

Article Title: APC is required for muscle stem cell proliferation and skeletal muscle tissue repair

doi: 10.1083/jcb.201501053

Figure Lengend Snippet: APC is required for adult skeletal muscle regeneration. (A) TM regimen and cardiotoxin (CTX) injury scheme for control and APC SC-KO mice. (B and C) Immunostaining for Pax7 (B) and e-MyHC (C) on TA cryosections 4 d after injury. (D–L) Analysis of TA muscles 14 d after injury reveals that tissue regeneration is defective in APC SC-KO muscles. (D) Whole sections of TA muscles immunostained for Laminin. Immunostaining for Tcf4 (F), type 1 Collagen (H), and Pax7 and Laminin (L) on TA cryosections is shown. Quantification of TA weight 14 d after injury normalized by uninjured TA weight (E), of the number of Tcf4+ cells/mm 2 (G), of relative Collagen+ area (I), and of sublaminar Pax7+ cells (normalized by Pax7+ cells of uninjured TA; J) is also shown. (K) GFP immunostaining on transverse sections of TAs isolated from Pax7 CreER ;Z/EG (Control-Z/EG) and Pax7 CreER APC lox/lox ;Z/EG (APC SC-KO-Z/EG) 14 d after injury demonstrates that APC-null satellite cells do not give rise to new myofibers, nor trans-differentiate in other cell types. Bars: (B, C, F, H, K, and L) 50 µm; (D) 200 µm. Nuclei are stained with Hoechst. Error bars indicate SEM. Inset panels show a 2× enlargement.

Article Snippet: Primary antibodies were as follows: mouse β-catenin (BD), goat Collagen Type I (SouthernBiotech), rabbit GFP (Life Technologies), rabbit Ki67 (Abcam), rabbit Laminin (Sigma-Aldrich), mouse MyoD (Dako), mouse embryonic-MyHC (Developmental Studies Hybridoma Bank), mouse Pax7 (DSHB), and rabbit Tcf4 (Cell Signaling Technology).

Techniques: Immunostaining, Isolation, Staining