Journal: bioRxiv

Article Title: Heterotypic inter-GPCR ß-arrestin coupling regulates lymphatic endothelial junctional architecture in murine lymph nodes

doi: 10.1101/435776

Figure Lengend Snippet: ( A ) Schematic of S1PR1 modulator screening system Four lentiviral vectors were transduced into U2OS cell line to enable gene activation by SAM and monitoring S1PR1 activation by TANGO system. The cells introduced with SAM sgRNA library were starved with 0.5% charcoal treated FBS, then the Venus-positive population was sorted and next-gen sequence (NGS) analysis was carried out to identify the enriched SAM sgRNA sequences. ( B ) Scatter plot showing enrichment of sgRNAs after sorting. Most sgRNAs are equally distributed in the pre-sort sample (closed gray circles) while after sorting a small fraction of sgRNAs (2,770 out of 70,290 sgRNAs) were enriched and others were not detected (open blue circles). The y-axis shows the NGS reads of sgRNAs. ( C ) Identification of top candidate genes using the MAGeCK method . The names of top ten candidate genes are indicated.

Article Snippet: The single clones were isolated from antibiotics resistant cells by limiting dilution, then introduced with the SAM sgRNA library (a gift from Feng Zhang, Addgene #1000000057) at a low multiplicity of infection.

Techniques: Activation Assay, Sequencing

Journal: Cell systems

Article Title: Illuminating Host-Mycobacterial Interactions with Genome-wide CRISPR Knockout and CRISPRi Screens.

doi: 10.1016/j.cels.2020.08.010

Figure Lengend Snippet: Figure 1. A Pooled Approach for CRISPR Knockout and CRISPRi Screening in Human THP-1 Cells (A) Strategy for preparing CRISPR libraries and performing genetic screens. (B) THP-1-mediated phagocytosis of M. bovis BCG after three rounds of infection (MOI 10:1) with induced green fluorescence (map24::GFP) (Scale bar, 20 mm). (C) Viability of host cells after three rounds of M. bovis BCG infection. (D and E) Expression of Cas9 (D) and dCas9-KRAB (E) in 9 randomly selected monoclonal THP-1 cells. Wild-type THP-1 cells were used as negative control. Vinculin was used as a loading control. (F) An sgRNA for EGFP was introduced in both wild-type and Cas9-expressing THP-1 cells using a lentivirus (pXPR-011) that also contains EGFP as a target (Scale bar, 20 mm). (G) Cas9-expressing THP-1 cells were transduced with an sgRNA targeting AAVS1 at a low MOI. Mutations at the AAVS1 locus were detected by SURVEYOR assay. The size of the AAVS1 amplicon is 500 bp. The cleaved product sizes are 320 and 180 bp. (H) Growth measurement associated with sgRNAs targeting INTS9, MCM2, and non-targeting negative controls sgNC1 and sgNC13. (I and J) RT-qPCR analysis of INTS9 (I) and MCM2 (J) expression in dCas9-KRAB-expressing THP-1 cells. The values are normalized to GAPDH (glyceraldehyde- 3-phosphate dehydrogenase). Data represent the mean ± SD (n = 3) (two-tailed unpaired Student’s t test, *p < 0.05 **p < 0.01 ***p < 0.001). See also Figure S1; Table S13.

Article Snippet: Human CRISPR knockout pooled library (Brunello) was obtained from Addgene (#73178).

Techniques: CRISPR, Knock-Out, Infection, Expressing, Negative Control, Control, Transduction, Amplification, Quantitative RT-PCR, Two Tailed Test

Journal: Cell systems

Article Title: Illuminating Host-Mycobacterial Interactions with Genome-wide CRISPR Knockout and CRISPRi Screens.

doi: 10.1016/j.cels.2020.08.010

Figure Lengend Snippet: Figure 2. Genome-wide Pooled CRISPR Knockout and CRISPRi Screens to Dissect Biological Pathways in Mycobacterial Infection (A and B) Volcano plots from CRISPR knockout (A) and CRISPRi (B) screens. For each sgRNA-targeted gene, the x axis shows its enrichment or depletion post- infection, and the y axis shows statistical significance measured by p value. Positive and negative screen hits are labeled as red and green dots, respectively. Gray dots represent non-targeting controls. For each screen, experiments were carried out in triplicate. (C) Enriched genes in the Venn diagram were filtered with a cut-off of FDR <0.1 and log2-fold change >1 in M. bovis BCG infection. The degree of significance of the overlap is given. (D) Gene-centric visualization of average fold change of CRISPR knockout and CRISPRi screens in infected versus non-infected host cells. Selected type I IFN and AHR/ARNT pathway components are highlighted in orange and blue. (E and F) Candidate genes identified by CRISPR knockout (E) and CRISPRi (F) screens were functionally categorized to understand the changes in biological functions involved in M. bovis BCG infection. Pathways shown in red are those identified by both screens. Color gradient of nodes represents the enrichment scores of gene sets. Node size represents the number of genes in the gene set. Edge width represents mutual overlap of genes. See also Figures S2 and S3; Tables S1, S2, S3, S4, S5, S11, and S12.

Article Snippet: Human CRISPR knockout pooled library (Brunello) was obtained from Addgene (#73178).

Techniques: Genome Wide, CRISPR, Knock-Out, Infection, Labeling

Journal: Cell systems

Article Title: Illuminating Host-Mycobacterial Interactions with Genome-wide CRISPR Knockout and CRISPRi Screens.

doi: 10.1016/j.cels.2020.08.010

Figure Lengend Snippet: Figure 3. Secondary CRISPR Knockout and CRISPRi Screens Identify Host Genetic Hits in Mycobacterial Infection (A) Enriched genes were filtered with a cut-off of FDR <0.05 and log2-fold change >0.5 in M. bovis BCG infection. The degree of significance of the overlap is given. (B) Validation rate of genetic hits in secondary screens grouped by their p values in primary genome-wide screens in M. bovis BCG infection. Number of genes per category is indicated. (C) Genetic hits from both primary and secondary screens were ranked by their differential sgRNA abundance between M. bovis BCG-infected versus uninfected populations (log2 fold change). (D) Heatmap of screen hits (log2 fold change) clustered in different biological pathways in M. bovis BCG infection. See also Figures S4 and S5; Tables S6, S7, S8, S9, and S10.

Article Snippet: Human CRISPR knockout pooled library (Brunello) was obtained from Addgene (#73178).

Techniques: CRISPR, Knock-Out, Infection, Biomarker Discovery, Genome Wide