Journal: Cell Reports Medicine

Article Title: Ongoing evolution of SARS-CoV-2 drives escape from mRNA vaccine-induced humoral immunity

doi: 10.1016/j.xcrm.2024.101850

Figure Lengend Snippet:

Article Snippet: pTwist-SARS-CoV-2 Spike-ΔC18 SA501.V2 (Triple RBD) R683G (GGC) , This study , Addgene Plasmid ID 212387.

Techniques: Virus, Recombinant, Saline, Modification, Plasmid Preparation, Software, Control

Journal: Innate Immunity

Article Title: A genome-wide CRISPR screen identifies regulation factors of the TLR3 signalling pathway

doi: 10.1177/1753425920915507

Figure Lengend Snippet: Outline of the genome-wide CRISPR/Cas9 forward genetic screen to identify genes required for TLR3 signalling. (a) Transduction of KBM7 cells with TLR3 cDNA leads to the expression of both full-length (*) and cleaved/mature (**) TLR3. Immunoblot of cell lysates of KBM7 cells complemented by the indicated cDNA was revealed by an anti-TLR3 mAb. As a loading control, tubulin was revealed with a specific mAb. (b) Design of the forward genetic screen in human near-haploid KBM7 cells. Creation of the NF-κB reporter KBM7 cell line by transduction of three independent expression cassettes encoding for dscGFP, TLR3 and Cas9. DscGFP expression was controlled by a NF-κB dependent promoter. Cells were transduced with the lentiviral GeCKO v2 sgRNA library (122,411 sgRNAs). Cells that were successfully transduced were selected with puromycin. After selection, the population was split into two; half was not sorted to represent the entire library, while the remaining population was sorted by flow cytometry after poly(I:C) stimulation to enrich for dscGFP negative cells. After three rounds of poly(I:C) stimulation/enrichment, the DNA from the enriched populations (rounds 1, 2 and 3) was harvested, and enriched sgRNAs were identified by sequencing and compared to an unsorted library. FACS plots obtained after each sort on dscGFP-negative cells show progressive enrichment rates of dscGFP-negative cells. (c) and (d) Proportion of cells responding to poly(I:C) as judged by dscGFP expression before and after sorting during the sequential enrichment process. Polyclonal or clonal cell populations were stimulated with poly(I:C; twice at 35 µg/ml, 4 h apart) or TNF-α (10 ng/ml) for 16 h before sorting at each round. In both cases, the proportion of dscGFP negative cells increased at each round of enrichment. sgRNA: single-guide RNA; NF-κB TRE: NF-κB transcription responsive element; min CMV: minimal CMV; dscGFP: destabilized copepod GFP; SFFV: spleen focus-forming virus promoter; 2A: peptide bond skipping sequence; BFP: blue fluorescent protein: PGK: phosphoglycerate kinase promoter; Hygro: hygromycin resistance gene.

Article Snippet: The MOI of the GeCKO v2 sgRNA library A and B (generously deposited by Prof. Feng Zhang ; #1000000047; Addgene) was estimated on cells exposed to various volumes of viruses and cultured with or without puromycin before counting the remaining cells.

Techniques: Genome Wide, CRISPR, Transduction, Expressing, Western Blot, Control, Selection, Flow Cytometry, Sequencing, Virus

Journal: Innate Immunity

Article Title: A genome-wide CRISPR screen identifies regulation factors of the TLR3 signalling pathway

doi: 10.1177/1753425920915507

Figure Lengend Snippet: Genome-wide screen of the TLR3 pathway identifies expected and new genes. (a) –log P value of the results of sgRNA enrichments identified by sequencing, corresponding to 42 genes. After one, two or three rounds of enrichment, the DNA from the enriched cell population was harvested, and enriched sgRNAs were identified by sequencing and comparison to an unsorted library. This determination was carried out for the two independent screens performed in parallel, and sequencing was performed twice generating a total of 12 enriched cell populations. Genes are presented when –log( P value) > 4 for at least 2/12 comparisons. Each dot represents the gene enrichments calculated from one condition. The RSA algorithm was used to identify the significantly enriched genes targeted in the selected cells. Red: known key members of TLR3 pathway; blue: further validated genes. (b) Plot illustrating the hits from the genetic screen. Mean log( P values) determined with the clonal population as a function of those found with polyclonal population. Each dot is a gene. Red squares: known key members of the TLR3 pathway; light blue triangles: genes further validated; grey dots: genes further tested; density colours: remaining genes out of the 42 found in panel (a).

Article Snippet: The MOI of the GeCKO v2 sgRNA library A and B (generously deposited by Prof. Feng Zhang ; #1000000047; Addgene) was estimated on cells exposed to various volumes of viruses and cultured with or without puromycin before counting the remaining cells.

Techniques: Genome Wide, Sequencing, Comparison

Journal: Innate Immunity

Article Title: A genome-wide CRISPR screen identifies regulation factors of the TLR3 signalling pathway

doi: 10.1177/1753425920915507

Figure Lengend Snippet: DLX1, SOD1 and AhR are required for a proper TLR3 response. (a) Heat map of the GFP expression results obtained with KBM7Rep cells transduced with the various sgRNAs ( n = 3 independent experiments). KBM7Rep cells transduced with the indicated sgRNAs were exposed to increasing doses of poly(I:C) or TNF-α (10 ng/ml) for 16 h before measurement of the GFP expression by FACS. The cluster of genes containing two sgRNAs targeting UNC93B1 and one targeting TLR3 is magnified. red: positive controls; blue: genes further studied; grey: negative controls. (b) NF-κB activity or (c) IL-8 and (d) IP-10 were measured by FACS or cytometric bead assay (CBA), respectively, in response to poly(I:C) exposure of the indicated cells (two-way ANOVA between one sgRNA condition and ctrl Cas9 ctrl). (e), (f) and (g) The indicated parameters were measured in the absence or presence of TNF-α ( n = 3 independent experiments). *, **, *** or ****: significantly different from ctrl Cas9 (Friedman test, with Dunn’s multiple comparison test between sgRNA conditions and Cas9 ctrl). UNC: UNC93B1. * P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001; **** P ≤ 0.0001.

Article Snippet: The MOI of the GeCKO v2 sgRNA library A and B (generously deposited by Prof. Feng Zhang ; #1000000047; Addgene) was estimated on cells exposed to various volumes of viruses and cultured with or without puromycin before counting the remaining cells.

Techniques: Expressing, Transduction, Activity Assay, Comparison

Journal: STAR Protocols

Article Title: Protocol for genome-scale in vivo CRISPR screening to study protection of beta cells under autoimmunity in a type 1 diabetes mouse model

doi: 10.1016/j.xpro.2023.102155

Figure Lengend Snippet:

Article Snippet: Mouse GeCKO-v2 (Genome-Scale CRISPR Knock-Out) Library A , Addgene , Cat# 1000000052.

Techniques: Virus, Recombinant, Red Blood Cell Lysis, Plasmid Preparation, CRISPR, Knock-Out, Software, Spectrophotometry, In Vivo Imaging, Transferring, Bioassay

Journal: PLoS Genetics

Article Title: Identification of the transcription factor Miz1 as an essential regulator of diphthamide biosynthesis using a CRISPR-mediated genome-wide screen

doi: 10.1371/journal.pgen.1009068

Figure Lengend Snippet: A . A workflow of the whole genome CRISPR knockout screen. 6 × 10 7 HT1080 (Cas9 expressing) cells were infected with the human lentiviral CRISPR library A or B (M.O.I. = 0.3). The CRISPR gene-edited cells were selected by puromycin (5 μg/ml) for 10 days. Then, the cells were treated with PA/FP59 (100 ng/ml each, about 10-fold of the minimum lethal concentration) twice with a two-day interval between treatments, and the toxin-resistant cells were allowed to form colonies by further culturing. The surviving, toxin-resistant cells were pooled, DNA extracted, and the sgRNA-containing fragments amplified and analyzed by Illumina deep sequencing for sgRNAs. B . Top hits from both library-A and library-B by Illumina deep sequencing. Only the genes with Log2(fold change) (LFC) ≥ 3 and were hit by ≥ 2 sgRNAs were considered as true positives and listed, along with the top four potential hits with just a single sgRNAs. Please refer to the and for details.

Article Snippet: The genome-wide CRISPR knockout lentiviral pooled human library-A and library-B developed by Feng Zhang’s MIT laboratory [ ] were used for our unbiased screens for host genes required for the toxin activity (Addgene Cat. # 1000000049).

Techniques: CRISPR, Knock-Out, Expressing, Infection, Concentration Assay, Amplification, Sequencing

Journal: PLoS Genetics

Article Title: Identification of the transcription factor Miz1 as an essential regulator of diphthamide biosynthesis using a CRISPR-mediated genome-wide screen

doi: 10.1371/journal.pgen.1009068

Figure Lengend Snippet: A - C . Knockout of Miz1 in HT1080 (A), RAW264.7 (B), and B16F10 (C) cells by CRISPR gene editing rendered these cells resistant to PA/FP59. The results from several independent clones are shown. In (A), knockout of Dph1 was used as an additional control. The indicated cells were incubated with various concentrations of PA in the presence of FP59 (100 ng/ml) for 48 h, followed by an MTT assay evaluating cell viability. Right panel in (C), lacking of Miz1 expression in representatives of B16 Miz1-KO clones was confirmed by Western blotting using an anti-Miz1 antibody (R&D Systems Cat. No. AF3760). Data are represented as mean (triplicate) ± SD. Unpaired two-tailed Student's t -tests, *, p < 0.001 between wild-type HT1080 (in A), RAW264.7 (in B), or B16F10 (in C) cells and their isogenic Miz1-KO or Dph1-KO cells. D and E . Reversion of the toxin-resistant phenotype of the B16 Miz1-KO cells (D) and HT1080 Miz1-KO cells (E) by transfection of a Miz1-expression plasmid. The results from several independent clones are shown. Cells were incubated with various concentrations of PA in the presence of FP59 (100 ng/ml) for 48 h, followed by an MTT assay evaluating cell viability. Of note, the Miz1-KO cells transfected with Miz1 regained their sensitivity to the toxin. Data are represented as mean (triplicate) ± SD. Unpaired two-tailed Student's t -tests, *, p < 0.001 between B16F10 Miz1-KO (in D) or HT1080 Miz1-KO (in E), and their isogenic Miz1 transfected cells.

Article Snippet: The genome-wide CRISPR knockout lentiviral pooled human library-A and library-B developed by Feng Zhang’s MIT laboratory [ ] were used for our unbiased screens for host genes required for the toxin activity (Addgene Cat. # 1000000049).

Techniques: Knock-Out, CRISPR, Clone Assay, Control, Incubation, MTT Assay, Expressing, Western Blot, Two Tailed Test, Transfection, Plasmid Preparation