human crispri pooled library (dolcetto)  (Broad Institute Inc)

Journal: Cell reports

Article Title: Requirements for the neurodevelopmental disorder-associated gene ZNF292 in human cortical interneuron development and function

doi: 10.1016/j.celrep.2025.115597

Figure Lengend Snippet: (A) Schematic illustrating methods for generating CRISPRi-KD pluripotent stem cell (hPSC) models by stable dCas9-KRAB expression without (control) versus with CROP-Opti-seq G1 or G2 gRNA expression plasmid. (B) Protocol used to differentiate hPSCs (day [D] 0) into NPCs (D15) and cINs (D30). (C and D) (C) gRNA (G1/G2) expression significantly reduced ZNF292 in hPSC-derived NPCs also expressing dCas9-KRAB (control), with (D) immunoblotting quantification (60% ± 7.5%-G1 and 55% ± 13.6%-G2 of control; n = 3 biological replicates; for all data see ). (E–H) Reduced outgrowth from NPC spheres (see ), with representative images and quantification at (E and F) D11 and (G and H) D15 ( n = 3). (I) Cell adhesion assay in D15 NPCs ( n = 4). (J) Cell count assay 48 h after seeding ( n = 3). (K) Principal-component analysis depicting variation in control and KD (G1/G2) NPC samples ( n = 4, each sample type). (L) Heatmap visualizing differential expression of the 2,267 common DEGs in G1/G2 KD versus control NPCs. (M and N) Gene ontology analysis of significant DEGs in KD versus control. Analysis in (E)–(J) by one-way ANOVA is represented as the mean ± SEM; * p < 0.05, ** p < 0.01, and **** p < 0.0001. Scale bars, 100 μm.

Article Snippet: Guide RNA: ZNF292 G1-Forward , Addgene: Broad GPP - Human CRISPRi Dolcetto Pooled Libraries , CACCGGAGCAGGAGAGGTTGAGTTG.

Techniques: Expressing, Control, Plasmid Preparation, Derivative Assay, Western Blot, Cell Adhesion Assay, Cell Counting, Quantitative Proteomics

Journal: Nature communications

Article Title: Whole-genome screens reveal regulators of differentiation state and context-dependent migration in human neutrophils.

doi: 10.1038/s41467-023-41452-x

Figure Lengend Snippet: Fig. 5 | Cell migration CRISPRi screen identifies genes important for adhesion and migration on 2D surfaces. a Left, components of inside-out αMβ2 integrin signaling. Right, normalized log2 fold-changes values for most significant sgRNA in the chemotaxis and chemokinesis screens. Error bars represent mean values +/− SEM across n = 28 measurements from 14 independent experiments. The gray shaded region shows the histogram of control sgRNAs. b Representative phase images of cell migration on fibronectin-coated coverslips (ITGB2 sgRNA and con- trol cells). Three fields of view were collected for each cell line. c Representative phase images of cell migration on fibronectin-coated coverslips (FLCN sgRNA, LAMTOR1 sgRNA, and control cells). Two fields of view were collected for each cell line. d Characterization of cell migration phenotypes. Speed was calculated by tracking cell nuclei during migration on fibronectin-coated coverslips. Persistence was inferred from the cell velocity data as described by Metzner et al. (see “Methods”). Measurements represent experiments performed over 2–3 days, acquired across 32 (sgControl), 10 (sgFLCN), and 14 (sgLAMTOR1) fields of view.

Article Snippet: The sgRNA library was previously reported in Sanson et al. (Dolcetto CRISPRi library set A, Addgene #92385).

Techniques: Migration, Chemotaxis Assay, Control

Journal: Nature communications

Article Title: Whole-genome screens reveal regulators of differentiation state and context-dependent migration in human neutrophils.

doi: 10.1038/s41467-023-41452-x

Figure Lengend Snippet: Fig. 6 | Cell migration CRISPRi screen identifies genes important for 3D amoeboid migration. a Comparison of normalized log2 fold-changes across the pooled CRISPRi cell migration screens of 3D amoeboid migration and chemokin- esis. b Comparison of 3D migration normalized log2 fold-changes with measure- ments of cell speed and migratory persistence via single-cell nuclei tracking. Cells from individual sgRNA knockdown lines were tracked during migration in collagen for 60 min (1 min frame rate). The median cell speed (left) and inferred migratory persistence (right, see “Methods”) are plotted against their measured normalized log2 fold-change from the pooled screen. Error bars represent mean values +/−SEM across independent experiments (6 screen replicates and 4 for experiments using

Article Snippet: The sgRNA library was previously reported in Sanson et al. (Dolcetto CRISPRi library set A, Addgene #92385).

Techniques: Migration, Comparison, Knockdown

Journal: Nature communications

Article Title: Whole-genome screens reveal regulators of differentiation state and context-dependent migration in human neutrophils.

doi: 10.1038/s41467-023-41452-x

Figure Lengend Snippet: Fig. 7 | Summary of pathways and genes identified across cell migration CRISPRi screens. a Pathways enriched in cell migration screens. Since the majority of gene knockdowns lead to poorer migratory phenotypes (i.e., negative log2 fold- changes), disruption of the noted pathways are associated with poorer migratory success. Due to the correlation across the chemotaxis and chemokinesis screens, their data was combined in this analysis (green). Pathways enriched in the 3D amoeboid migration screen are shown in yellow. p values estimate the statistical significance of gene set enrichment, calculated using a one-sided permutation test and adjusted for multiple comparisons using the Benjamini–Hochberg procedure.

Article Snippet: The sgRNA library was previously reported in Sanson et al. (Dolcetto CRISPRi library set A, Addgene #92385).

Techniques: Migration, Disruption, Chemotaxis Assay

Journal: bioRxiv

Article Title: Cell migration CRISPRi screens in human neutrophils reveal regulators of context-dependent migration and differentiation state

doi: 10.1101/2022.12.16.520717

Figure Lengend Snippet: (A) CRISPRi gene knockdown using sgRNA targeting the CD4 gene in uHL-60 cells. Flow cytometry immunofluorescence of CD4 protein expression shows near-complete loss of protein (blue) when compared to isotype antibody control (gray, shaded), representing cellular autofluorescence. Normal expression of CD4 in uHL-60 cells is shown in orange. (B) Schematic of pooled CRISPRi dropout experiments of proliferation of uHL-60 cells and differentiation into dHL-60 neutrophils. A pooled sgRNA library was integrated into uHL-60 cells expressing dCas9-KRAB using lentiviral transduction and selected for stable integration with puromycin for seven days. Proliferation was assayed by comparing sgRNA abundances following six days of growth (∼24 hr doubling time) (set 2 versus set 1). Differentiation was assayed by comparing sgRNA distribution in dHL-60 neutrophils with uHL-60 cells (set 3 versus set 1). The proliferation screen was performed four times, while the differentiation screen was performed eight times. (C) Schematic of pooled CRISPRi cell migration assays. Migration in dHL-60 cells was assayed across three experiments: The first two assessed migration through track-etch membranes with 3 μm diameter pores, stimulating migration either through the presence of a serum gradient (chemotaxis, 10% hiFBS added to the bottom reservoir) or in a uniform serum environment (chemokinesis, uniform 10% hiFBS added throughout media). The third assay assessed migration through an extracellular matrix composed of collagen and fibrin (see Methods for additional details). For quantification of migration screens, sgRNA abundance in both migratory fractions (sets 4i and 5i) and remaining cells (sets 4ii and 5ii) were compared to our initial dHL-60 library (set 3). Membrane, pores and cells drawn to scale. (D) Quantification of the migratory cell populations collected in the different migratory screens (i.e. sets 4ii and 5ii). Migration through 3 μm diameter pores of track-etch membranes was assayed at two time points (2 hr, 6 hr) and (+/-) a gradient of hiFBS. Cells were collected from the extracellular matrix after nine hours. Error bars represent standard deviation (amoeboid, 3D: N = 6; chemokinesis: 2 hr, N = 16, 6 hr, N = 12; chemotaxis: 2 hr and 6 hr, N = 4). (E) Volcano plots showing the statistical significance across the CRISPRi screens of proliferation, differentiation, and cell migration. Data points represent the average log 2 fold-change across the three sgRNA per gene. Cell migration values represent an average across all migration assays. Control data points were generated by randomly selecting groups of three control sgRNAs. Adjusted p-values were calculated using permutation tests with the dashed line representing a value of 0.05. See Methods for additional details on data analysis and number of replicates for each experiment. (F) Histogram plots show the number of significant genes using an adjusted p-value cutoff of 0.05 (left horizontal bar plot) and the intersection of genes across each of the screens (vertical bar plot). The dot diagram identifies the specific screens considered for each intersection in the vertical bar plot.

Article Snippet: The sgRNA library was previously reported in Sanson et al. 2018 (Dolcetto CRISPRi library set A, Addgene #92385).

Techniques: Knockdown, Flow Cytometry, Immunofluorescence, Expressing, Control, Transduction, Migration, Chemotaxis Assay, Membrane, Standard Deviation, Generated

Journal: bioRxiv

Article Title: Cell migration CRISPRi screens in human neutrophils reveal regulators of context-dependent migration and differentiation state

doi: 10.1101/2022.12.16.520717

Figure Lengend Snippet: (A) Pathways enriched in our CRISPRi differentiation screen (dHL-60 cells relative to uHL-60 cells). Pathways that were associated with genes whose knockdown predominantly led to an enrichment of target sgRNAs in the dHL-60 cells are identified in blue, while those that decreased in abundance are in red. (B) Comparison of log 2 fold-changes across the CRISPRi screens of proliferation, differentiation, and cell migration. Several gene sets identified through our pathway or known regulators of neutrophil differentiation are identified. (C) Schematic of mTORC1/ mTORC2 signaling pathway, color coded by signed statistical significance values (log 10 p adj -value) from the differentiation screen results. Blue indicates gene targets whose sgRNA were enriched in the dHL-60 cells, while red indicates those that were depleted. (D) Differentiation screen results were confirmed across a number of sgRNA targets. Cell density was monitored at 5-days following the initiation of neutrophil differentiation. The dashed lines represent the values obtained for dHL-60 cells with a control sgRNA. Error bars represent standard deviation across replicates (N=8 for screen, N=3 cell density). (E) Change in dHL-60 cell density following differentiation. Note that media was replenished every three days, with cell density measurements corrected to account for changes in media volume and evaporation. Cell density measurements were normalized to day 4, following initiation of cell differentiation of uHL-60 cells. Error bars represent standard deviation across replicates (N=3).

Article Snippet: The sgRNA library was previously reported in Sanson et al. 2018 (Dolcetto CRISPRi library set A, Addgene #92385).

Techniques: Knockdown, Comparison, Migration, Control, Standard Deviation, Evaporation, Cell Differentiation

Journal: bioRxiv

Article Title: Cell migration CRISPRi screens in human neutrophils reveal regulators of context-dependent migration and differentiation state

doi: 10.1101/2022.12.16.520717

Figure Lengend Snippet: (A) Comparison of normalized log 2 fold-changes across the pooled CRISPRi cell migration screens of chemotaxis and chemokinesis. Genes associated with inside-out α M β 2 integrin signaling are among most significantly enriched across both assays, with ITGB2, FERMT3 and TLN1 genes identified. (B) Schematic of the key components of inside-out α M β 2 integrin signaling and summary of their most significant sgRNA normalized log 2 fold-changes values, averaged across all the chemotaxis and chemokinesis screens. Error bars represent standard error of the mean across individual screen experiments (N = 14). The gray shaded region shows the histogram of control sgRNAs. (C) Phase microscopy of cell migration on fibronectin-coated coverslips with dHL-60 CIRSPRi knockdown lines targeting ITGB2 and a control sgRNA. The elongated morphology of the ITGB2 knockdown line is because the cell is detached from the coverslip. Time courses are shown in Figure S4. (D) Comparison of chemotaxis normalized log 2 fold-changes with measurements in individual sgRNA knockdown lines. Individual cell lines were exposed to a serum gradient by adding 10% hiFBS to the bottom-side of the track-etch membrane and allowing cells to migrate for two hours. The gray data point and dashed lines represent the values obtained for cells with a control sgRNA. Error bars represent standard error of the mean across replicates (N = 4 for individual transwell experiments using single sgRNAs). (E) Brightfield microscopy of uHL-60 CIRSPRi knockdown lines targeting ATIC and a control sgRNA. Control uHL-60 cells exhibit an expected round morphology, while sgRNA targeting of ATIC resulted in many cells that exhibited a migratory capability (white arrows). (F) Characterization of cell migration phenotypes following knockdown of GIT2. Speed was calculated by tracking cell nuclei during migration on fibronectin-coated coverslips. Persistence was inferred from the cell velocity data as described by Metzner et al. (see Methods ). Individual data points represent average values for cells across a single field of view, with the shaded regions showing the distribution of all measurements. Measurements represent experiments performed over 3 days.

Article Snippet: The sgRNA library was previously reported in Sanson et al. 2018 (Dolcetto CRISPRi library set A, Addgene #92385).

Techniques: Comparison, Migration, Chemotaxis Assay, Control, Microscopy, Knockdown, Membrane

Journal: bioRxiv

Article Title: Cell migration CRISPRi screens in human neutrophils reveal regulators of context-dependent migration and differentiation state

doi: 10.1101/2022.12.16.520717

Figure Lengend Snippet: (A) Comparison of normalized log 2 fold-changes across the pooled CRISPRi cell migration screens of amoeboid, 3D migration and chemokinesis. (B) Comparison of amoeboid 3D normalized log 2 fold-changes with measurements of cell speed and migratory persistence via single-cell nuclei tracking. Cells from individual sgRNA knockdown lines were tracked during migration in collagen for 60 minutes (1 minute frame rate). The median cell speed (left) and inferred cell persistence (right, see methods) are plotted against their measured normalized log 2 fold-change from the pooled screen. Error bars represent standard error of the mean across replicate experiments (N = 6 for screens and N= 4 for experiments using single sgRNA cell lines). (C) & (D) show immunofluorescence localization of FMNL1 and CORO1A in amoeboid-migrating cells in collagen. F-actin was labeled by phalloidin, while DNA was stained by DAPI. Images are maximum projections; right panels show grayscale localization of formin-like 1 (C) and coronin 1A (D). Red arrows indicate the direction of cell migration.

Article Snippet: The sgRNA library was previously reported in Sanson et al. 2018 (Dolcetto CRISPRi library set A, Addgene #92385).

Techniques: Comparison, Migration, Knockdown, Immunofluorescence, Labeling, Staining

Journal: Life Science Alliance

Article Title: CRISPRi screening reveals regulators of tau pathology shared between exosomal and vesicle-free tau

doi: 10.26508/lsa.202201689

Figure Lengend Snippet: (A) Western blot analysis of sarkosyl-soluble and sarkosyl-insoluble fractions obtained from brains of P301L tau transgenic rTg4510 mice. The insoluble fraction exhibits phosphorylated tau (pS422) and high molecular weight total tau (Tau5). (B, C) Epifluorescence microscopy detecting tau RD-YFP in tau biosensor cells 48 h after treatment with sarkosyl-soluble (B) and sarkosyl-insoluble tau (C). Brighter spots (arrowheads) representing tau aggregates only appear in cells that were treated with sarkosyl-insoluble tau. Scale bar: 50 µm. (D) Schematic representation of the pooled CRISPRi screen. Tau biosensor cells expressing the FRET pair tau RD-CFP and tau RD-YFP together with lentiviral KRAB-dCas9 (BSKRAB) were transduced with the Dolcetto CRISPRi library containing pooled lentiviral sgRNAs targeting ∼18,000 genes, followed by incubation with sarkosyl-insoluble tau (vesicle-free tau seeds). 48 h later, the cells were sorted into FRET(+) and FRET(−) populations using FACS. Samples were processed to generate an NGS sequencing library and sequenced on a NextSeq 500 instrument. (E) Frequency distribution of mean sgRNA read counts across all samples. 39 out of 57,050 sgRNAs (0.068%) were not detected. (F) Volcano plot showing genewise log 2 fold changes of sgRNA counts versus false discovery rate. False discovery rate values were based on robust ranking aggregation from MAGeCK . Genes that were followed up are highlighted in red. (G) Gene ontology-term enrichment analysis of the top 200 positive regulators of tau aggregation identified by CRISPRi screening and enrichment for relevant pathways.

Article Snippet: Human CRISPRi sgRNA library Dolcetto Set A ( ) (#92385; Addgene) was transformed into electrocompetent Lucigen Endura Escherichia coli (60242-2; Lucigen) using program EC1 on MicroPulser Electroporator (1652100; Bio-Rad) following the manufacturer’s instructions.

Techniques: Western Blot, Transgenic Assay, High Molecular Weight, Epifluorescence Microscopy, Expressing, Transduction, Incubation, Sequencing

Journal: Life Science Alliance

Article Title: CRISPRi screening reveals regulators of tau pathology shared between exosomal and vesicle-free tau

doi: 10.26508/lsa.202201689

Figure Lengend Snippet: (A) Schematic representation of the workflow for functional validation. Individual sgRNAs were used to silence the corresponding genes in combination with KRAB-dCas9 in tau biosensor cells (BSKRAB-KD), then treated with either sarkosyl-insoluble tau (vesicle-free tau seeds) or exosome-like EVs for 72 h, followed by detection and quantification of tau aggregation using FRET flow cytometry. A fraction of the same BSKRAB-KD cells was also grown for 72 h to corroborate the knockdown of protein expression using Western blots. (B, C, D, E, F, G) Integrated FRET intensities represent levels of tau aggregation upon knocking down the different targets. The control black column (NT) is the average obtained with three independent non-targeting sgRNAs (n = 3) assessed in triplicate. Control cells were compared with knockdown cells targeted individually (1, 2, and 3). Error bars represent the SEM for n = 3, * P < 0.05; ** P < 0.01; *** P < 0.001; and **** P < 0.0001. Each single targeting sgRNA increased tau aggregation with both exosomal and vesicle-free tau seeds. (C, D) Interestingly, ANKLE2 and BANF1 (C, D) appear to induce a stronger effect on tau aggregation induced by exosome-like EVs. (H) Quantitative Western blot analysis of BSKRAB-KD knockdown cells. Each sgRNA generated a protein knockdown of the targeted gene. Note that the ANKLE2-specific antibody reacted with several isoforms, including the canonical variant sized 104–117 kD (red box outline); however, all isoforms were down-regulated when the ANKLE2 locus was silenced. Similarly, the EIF1AD antibody recognized the canonical isoform of 19 kD and one additional variant of lower molecular weight, both being silenced with the individual sgRNA against EIF1AD. (I) Quantification of the extent of protein knockdown for the different targeted genes. Error bars represent the SEM for n = 3, * P < 0.05; ** P < 0.01; and **** P < 0.0001.

Article Snippet: Human CRISPRi sgRNA library Dolcetto Set A ( ) (#92385; Addgene) was transformed into electrocompetent Lucigen Endura Escherichia coli (60242-2; Lucigen) using program EC1 on MicroPulser Electroporator (1652100; Bio-Rad) following the manufacturer’s instructions.

Techniques: Functional Assay, Biomarker Discovery, Flow Cytometry, Knockdown, Expressing, Western Blot, Control, Generated, Variant Assay, Molecular Weight

Journal: Life Science Alliance

Article Title: CRISPRi screening reveals regulators of tau pathology shared between exosomal and vesicle-free tau

doi: 10.26508/lsa.202201689

Figure Lengend Snippet: (A) FACS plots of knockdown and control cells (non-targeting sgRNA) without adding exogenous tau seeds. Quadrants (Q2 shaded in yellow) in which FRET-positive cells were detected revealed the absence of a FRET signal in both control (NonT) and knockdown cells for ANKLE2, BANF1, VPS18, EIF1AD, and NUSAP1, indicating that no spontaneous tau aggregation was initiated. (B) However, these cells (bottom panels) showed FRET-positive cells only after tau seeds (400 ng of sarkosyl tau) were added, implying the requirement for an exogenous tau seed to trigger the aggregation of endogenous tau. Percentages of FRET-positive cells in Q2 are shown (n = 3, average ± SEM, 40,000 cells/experiment were analyzed).

Article Snippet: Human CRISPRi sgRNA library Dolcetto Set A ( ) (#92385; Addgene) was transformed into electrocompetent Lucigen Endura Escherichia coli (60242-2; Lucigen) using program EC1 on MicroPulser Electroporator (1652100; Bio-Rad) following the manufacturer’s instructions.

Techniques: Knockdown, Control

Journal: mBio

Article Title: High-Throughput CRISPR Screens To Dissect Macrophage- Shigella Interactions

doi: 10.1128/mBio.02158-21

Figure Lengend Snippet: Genome-wide pooled CRISPR knockout and CRISPRi screens to dissect biological pathways in S. flexneri infection. (A) Monoclonal Cas9 and dCas9-Krab-expressing THP-1 cell lines were constructed and transduced with lentiviral sgRNA libraries. High-coverage CRISPR knockout and CRISPRi libraries were split for subsequent S. flexneri Δ virG infection. Uninfected host cells were collected for genomic DNA extraction immediately after the split. Surviving THP-1 cells with sgRNA barcodes were harvested until the total number reached >500-fold sgRNA coverage of screen libraries (2 to 3 weeks) and processed for next-generation sequencing. Genome-wide genetic hits were identified by comparing sgRNA abundances between infected samples and noninfected controls. Based on those host targets, secondary CRISPR knockout and CRISPRi screen libraries were designed and prepared. Similarly, host cell survival-based secondary positive screens were performed to validate those host targets. Finally, drug inhibitors that selectively inhibit genetic hits were tested. KO, knockout; KD, knockdown. (B and C) Volcano plots from genome-wide CRISPR knockout (B) and CRISPRi (C) screens. For each sgRNA-targeted gene, the x axis shows its enrichment (positive hits) or depletion (negative hits) postinfection, and the y axis shows statistical significance measured by P value. The top 3 positive and negative screen hits are labeled as red and green dots, respectively; positive hits were those that extended the survival of the THP-1 cells beyond 2 to 3 h of bacterial infection and negative hits those that shortened THP-1 cell survival. Gray dots represent nontargeting controls. For each screen, experiments were carried out in triplicate. (D and E) Genes identified by genome-wide CRISPR knockout (D) and CRISPRi (E) screens were functionally categorized to understand the biological functions involved in S. flexneri infection. The color gradient of nodes represents the enrichment scores of gene sets. Node size represents the number of genes in the gene set.

Article Snippet: The Dolcetto human CRISPRi pooled library was a gift from John Doench (the Broad Institute, also available from Addgene [catalog number 92385]).

Techniques: Genome Wide, CRISPR, Knock-Out, Infection, Expressing, Construct, Transduction, DNA Extraction, Next-Generation Sequencing, Knockdown, Labeling

Journal: mBio

Article Title: High-Throughput CRISPR Screens To Dissect Macrophage- Shigella Interactions

doi: 10.1128/mBio.02158-21

Figure Lengend Snippet: Genome-wide CRISPR knockout and CRISPRi screens to dissect enriched genes and biological pathways in S. flexneri infection. (A) Enriched genes in the Venn diagram were filtered with a cutoff FDR of <0.25 and log 2 fold change of >1 in S. flexneri Δ virG infection. The degree of significance of the overlap between genome-wide CRISPR knockout and CRISPRi screens is given. (B) Gene-centric visualization of the average log 2 fold change of CRISPR knockout and CRISPRi screens in S. flexneri -infected versus noninfected host cells. Selected components of TLR1/2, the pyruvate catabolism signaling pathway, and inflammasome formation are highlighted in orange, purple, and green, respectively. (C) Top enriched genes and associated biological pathways in S. flexneri Δ virG infection. The color gradient of gene boxes represents the log 2 fold change of gene sets in genome-wide CRISPR knockout and CRISPRi screens. TCA, tricarboxylic acid.

Article Snippet: The Dolcetto human CRISPRi pooled library was a gift from John Doench (the Broad Institute, also available from Addgene [catalog number 92385]).

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

Journal: mBio

Article Title: High-Throughput CRISPR Screens To Dissect Macrophage- Shigella Interactions

doi: 10.1128/mBio.02158-21

Figure Lengend Snippet: Secondary CRISPR knockout and CRISPRi screens identify host genetic hits in S. flexneri infection. (A) Enriched genes were filtered with a cutoff FDR of <0.05 and a log 2 fold change of >0.5 in S. flexneri Δ virG infection. The degree of significance of the overlap is given. (B) Validation rates of genetic hits in the secondary screen grouped by their P values in the genome-wide screens in S. flexneri Δ virG infection. The number of genes per category is indicated. (C) Genetic hits from both primary genome-wide and secondary screens were ranked by their differential sgRNA abundances between S. flexneri -infected and uninfected populations (log 2 fold change). (D) Heatmap of screen hits clustered in different biological pathways in S. flexneri Δ virG infection. ETC, electron transport chain; ROS, reactive oxygen species; NOD, nucleotide binding oligomerization domain.

Article Snippet: The Dolcetto human CRISPRi pooled library was a gift from John Doench (the Broad Institute, also available from Addgene [catalog number 92385]).

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

Journal: mBio

Article Title: High-Throughput CRISPR Screens To Dissect Macrophage- Shigella Interactions

doi: 10.1128/mBio.02158-21

Figure Lengend Snippet: Validation of top genetic hits and effects of the IRAK1 inhibitor in S. flexneri infection of human THP-1 cells. (A) Correlation between pooled screen and validation data. For each hit, the log 2 fold change obtained from the genome-wide CRISPRi screening data (screen phenotype) was plotted against the fold change of cell viability of genetic hits from levels in the nontargeting control cells (validation phenotype). Host cell viability was measured by trypan blue staining. sgNC80 and sgNC135 are nontargeting controls. R is the Pearson correlation coefficient. (B) Intracellular S. flexneri Δ virG level after infection of individual knockdown THP-1 cells, which was measured by counting bacterial CFU. (C) Schematic of positive genetic hits in the TLR1/2 signaling pathway and corresponding inhibitors. (D) Cytokine and chemokine production in infected THP-1 cells with MYD88 and IRAK1 knockdown. (E and F) Viability of THP-1 cells (E) and growth of the intracellular S. flexneri Δ virG mutant (F) postinfection in the presence or absence of the IRAK1 inhibitor IRAK1/4-Inh at different concentrations. Data represent the means ± standard deviations (SD) ( n = 3) (Student's two-tailed unpaired t test, * , P < 0.05; * * , P < 0.01; ** * , P < 0.001; ns, not significant).

Article Snippet: The Dolcetto human CRISPRi pooled library was a gift from John Doench (the Broad Institute, also available from Addgene [catalog number 92385]).

Techniques: Biomarker Discovery, Infection, Genome Wide, Control, Staining, Knockdown, Mutagenesis, Two Tailed Test