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

Journal: Nature Biomedical Engineering

Article Title: Arrayed CRISPR libraries for the genome-wide activation, deletion and silencing of human protein-coding genes

doi: 10.1038/s41551-024-01278-4

Figure Lengend Snippet: a , Cloning strategy. The ampicillin resistance gene (AmpR) was removed from the vector pYJA5. sgRNA1–4 and the trimethoprim resistance gene (TmpR) were fused with three distinct PCR amplicons. All elements were Gibson-assembled to form the qgRNA-pYJA5 plasmid, and transformants were selected with trimethoprim. The detailed structure of qgRNA-pYJA5 full plasmid and qgRNA cassette are depicted. LTR, long terminal repeat; Ψ, packaging signal sequence; PB, piggyBac transposon element; PuroR, puromycin resistance element; hU6, mU6, hH1 and h7SK are ubiquitously expressed RNA polymerase-III promoters; sg, sgRNA. F and R arrows: forward and reverse primers used for single-colony PCRs, Sanger and NGS. b , Representative pYJA5 restriction fragments, 3-fragment PCRs and single-colony PCR of ALPA cloning products after transforming into E. coli and trimethoprim selection. Bbs I digestion of pYJA5 yielded an ~1 kb band of the AmpR element and ~7.6 kb band of the linearized vector (left). After PCR with the corresponding sgRNA primers, the three amplicons showed the expected size of 761 bp, 360 bp and 422 bp on agarose gels, respectively (middle). Single-colony PCR with primers flanking the qgRNA cassette of ALPA cloning products in transformed bacteria plate consistently yielded the expected size (2.2 kb, right). c , Percentage of correct, recombined and mutated qgRNA plasmids in 8 independent ALPA cloning experiments with distinct qgRNA sequences (≥22 colonies were tested in each experiment). d , Percentage of correct, recombined and mutated qgRNA plasmids in four ALPA cloning experiments. Each dot represents an independent biological replicate consisting of eight colonies ( n = 24; mean ± s.e.m.). e , Timeline of ALPA cloning in high-throughput format (h, hours; d, days). Created with BioRender.com . f , Gene activation (qRT-PCR) in HEK293 cells 3 days post-transfection with dCas9-VPR and single (sg1–4) or four sgRNA (qg) plasmids. Additional genes are shown in Extended Data Fig. . Dots (here and henceforth): independent experiments (mean ± s.e.m.). g , Gene ablation efficiency by single sgRNAs versus qgRNAs in HEK293 cells via co-transfection with the Cas9 plasmid. 12 single sgRNAs (sg1–12) from the Brunello, GeCKOv2 and TKOv3 libraries were tested; qgRNA plasmids (qg-A,B,C,D) were assembled with the random combination of sg1–4, sg5–8 and sg9–12, and the 4 least effective single sgRNAs among the 12 sgRNAs, respectively. Outcomes were re-plotted for the four least effective single sgRNAs along with the respective qg-D. hNTo, NT control plasmid; cell-surface proteins were stained with fluorescent-conjugated antibodies and analysed via flow cytometry. h , qgRNA plasmids robustly ablated genes inadequately disrupted by single sgRNAs. Single sgRNAs were assembled into qgRNA plasmids and co-transfected with the Cas9 plasmid into HEK293 cells (as in g ). In f and g , P values were determined by one-way ANOVA with Dunnett’s multiple comparisons test.

Article Snippet: The accumulation of GFP-tagged SQSTM1 provides a reliable proxy of autophagic activity, motivating us to compare the sensitivity and specificity of a pooled T.spiezzo version with the two pooled CRISPRko libraries, Brunello and Cellecta.

Techniques: Cloning, Plasmid Preparation, Sequencing, Selection, Transformation Assay, Bacteria, High Throughput Screening Assay, Activation Assay, Quantitative RT-PCR, Transfection, Cotransfection, Control, Staining, Flow Cytometry

Journal: Nature Biomedical Engineering

Article Title: Arrayed CRISPR libraries for the genome-wide activation, deletion and silencing of human protein-coding genes

doi: 10.1038/s41551-024-01278-4

Figure Lengend Snippet: a , H4-Cas9-GFP-SQSTM1 cells were transduced with genome-wide pooled lentiviral sgRNA ablation libraries (T.spiezzo and Brunello) and selected with puromycin. Cells in the uppermost and lowermost fluorescence quartile were collected by FACS and sgRNAs were quantified by sequencing genomic DNA. Created with BioRender.com . b , c , Average log 2 FC in GFP high and GFP low samples from T.spiezzo versus Brunello ( b ) and T.spiezzo versus Cellecta ( c ). Autophagy-relevant genes (autophagy, GO:0006914 ) are highlighted in red. d , Autophagy genes enriched in GFP high cells from the T.spiezzo, Brunello and Cellecta screens. The box plot represents the interquartile range. e , Heatmap showing the highest and lowest mean log 2 FC (GFP high versus GFP low ) of genes identified among the top 100 genes in the T.spiezzo, Brunello and Cellecta screens. f , Quantification of log 2 FC of cell count in GFP high versus GFP low populations transduced with T.spiezzo qgRNA lentivirus against NT control or each of the 16 genes selected for validation. The boundaries for GFP high and GFP low cell populations were set according to the NT condition. Dots (here and henceforth): independent experiments, mean ± s.e.m. P values were determined by one-way ANOVA with Dunnett’s multiple comparisons test. g , An example of GFP-SQSTM1 puncta in H4-Cas9-GFP-SQSTM1 cells transduced with T.spiezzo qgRNA lentivirus against NT or HNRNPM (see Extended Data Fig. for all genes tested). Dashed lines: cell contours according to the cytosolic GFP signal. h , Percentage of cells with GFP-SQSTM1 puncta (purple) and puncta area (black) in H4-Cas9-GFP-SQSTM1 cells transduced with T.spiezzo qgRNA lentivirus against 16 genes selected for validation. P values were determined by one-way ANOVA with Dunnett’s multiple comparisons test, *** P < 0.001. i , Immunoblotting of LC3-II from H4-Cas9 cells transduced with T.spiezzo qgRNA lentivirus against 10 possible autophagy modulators in the absence (−) or presence (+) of ChQ (100 µM, 6 h). GAPDH: loading control. Two biological repeats were assessed for each condition. j , Representative micrographs of YFP-LC3 in H4-Cas9-cells transduced with T.spiezzo qgRNA lentivirus against NT or each of the 5 genes selected for further validation. H4-Cas9 cells were cultured and treated as described in a and transduced with YFP-LC3 lentiviruses 48–60 h before examining YFP-LC3 puncta. k , Quantification of puncta area of YFP-LC3 of cells and conditions described in j . N = 3 biological repeats. P values were determined by a one-tailed Mann–Whitney test.

Article Snippet: The accumulation of GFP-tagged SQSTM1 provides a reliable proxy of autophagic activity, motivating us to compare the sensitivity and specificity of a pooled T.spiezzo version with the two pooled CRISPRko libraries, Brunello and Cellecta.

Techniques: Transduction, Genome Wide, Fluorescence, Sequencing, Cell Counting, Control, Biomarker Discovery, Western Blot, Cell Culture, One-tailed Test, MANN-WHITNEY

Journal: Nature Biomedical Engineering

Article Title: Arrayed CRISPR libraries for the genome-wide activation, deletion and silencing of human protein-coding genes

doi: 10.1038/s41551-024-01278-4

Figure Lengend Snippet: a , Histogram showing the gating strategy to isolate GFP high and GFP low (upper and lower quartile of GFP fluorescence, respectively) cell populations. b , Percentages of sequencing reads in GFP high , GFP low , and unsorted samples from the T.spiezzo pooled screen that correctly aligned to sgRNA2 (mapped reads 1) or sgRNA3 (mapped reads 2), that did not align to sgRNA2 (unmapped reads 1) or sgRNA3 (unmapped reads 2) and those that aligned and had the correct linkage between sgRNA2 and sgRNA3 (mapped and linked). c - e , Overrepresentation analysis of the top 200 genes enriched in GFP high cell populations from the T.spiezzo ( c ), Brunello ( d ), and Cellecta ( e ) screens. Gene counts and adjusted p-value are represented in each figure. The 10 most significant GO biological processes are shown. f-m , Autophagy-related gene sets including autophagosome assembly ( GO:0000045 , n=174, f ), autophagosome membrane ( GO:0000421 , n=129, g ), autophagy of mitochondrion ( GO:0000422 , n=109, h ), autophagosome ( GO:0005776 , n=198, i ), regulation of autophagy ( GO:0010506 , n=209, j ), positive regulation of autophagy ( GO:0010508 , n=196, k ), macroautophagy ( GO:0016236 , n=180, l ), and lysosomal microautophagy ( GO:0016237 , n=6, m ) using absolute log 2 fold changes in GFP high cell populations from the T.spiezzo, Brunello, and Cellecta screens. The p value was determined by two-way ANOVA. The box plot represents the interquartile range. n , An example of flow cytometry histograms of GFP-SQSTM1 intensity in H4-Cas9-GFP-SQSTM1 cells transduced with T.spiezzo qgRNA lentivirus against each of the 16 genes selected for validation or a non-targeting control (NT) lentivirus. N = 3 biological repeats. o , An example of GFP-SQSTM1 puncta in H4-Cas9-GFP-SQSTM1 cells transduced with T.spiezzo qgRNA lentivirus against each of the 16 genes selected for validation or NT controls. N = 3 biological repeats. Cells were demarcated by dashed lines according to the cytosolic GFP signal. p , Quantification of LC3II levels of cells and conditions described in Fig. . All values were normalized to the mean of the two NT repeats (- ChQ) on the same blot. Both the LC3II and normalized LC3II (LC3II/GAPDH) levels were shown to determine whether consistent changes were observed for the two biological repeats of a defined gene to determine promising candidates for further validation.

Article Snippet: The accumulation of GFP-tagged SQSTM1 provides a reliable proxy of autophagic activity, motivating us to compare the sensitivity and specificity of a pooled T.spiezzo version with the two pooled CRISPRko libraries, Brunello and Cellecta.

Techniques: Fluorescence, Sequencing, Membrane, Flow Cytometry, Transduction, Biomarker Discovery, Control

Journal: PLoS ONE

Article Title: Considerations and practical implications of performing a phenotypic CRISPR/Cas survival screen

doi: 10.1371/journal.pone.0263262

Figure Lengend Snippet: (A) Viability staining of DIE cells treated with doxycycline (Doxy) concentrations (100, 250, 500 or 1000ng/ml) and for different exposure times (2, 4, 6, or 8h) to determine the optimal concentration and exposure time to induce sufficient cell death rates in DIE cells. Green circles indicate which conditions (low Doxy: 250ng/ml high Doxy: 1000ng/ml); were used for the genome-wide CRIPSR/Cas9 screen. (B) The CRISPR/Cas9 screen timeline from the time of library transfection (Day 0) to the final harvest of surviving DIE cells (Day 10). 6 days after transfection of the library, Doxycycline (Doxy) was added for 24h to induce DUX4 expression. Low Doxy: 250ng/ml; high Doxy: 1000ng/ml; early harvest: 24h after Doxy removal; late harvest: 48h after Doxy removal. (C) Volcano plot showing the enrichment of sets of guides of the low doxycycline 250ng/ml) -early harvest (24h after doxycycline removal) screen (early-low). Blue points represent guide sets that are significantly enriched (P-value ≤ 0.01), LFC ≥ 1), green points are the positive controls (DUX4, MAST1, MGAT4B), red points represent the non-target/negative control guides. (D) Chromosomal ideogram indicating the location of enriched hits in the human genome, of the low doxycycline-early harvest screen (see panel B). (E) Schematic representation of the location of a small number of false positive hits on chromosome 5 and chromosome 19. (F) Viability staining demonstrating surviving DIE-Cas9 cells (DIE cells constitutively expressing Cas9) after 250ng/ml doxycycline exposure, containing knockouts of the same genes mentioned in (E), but also DUX4, MGAT4B and MAST1. Media did not contain any selection markers (blasticidin or puromycin) to select for the presence of the rtTA3 or the DUX4 transgene. NT: Non-target controls.

Article Snippet: The Human Brunello CRISPR knockout pooled lentiviral prep library was a gift from David Root and John Doench (Broad Institute, MA, U.S.A.).

Techniques: Staining, Concentration Assay, Genome Wide, CRISPR, Transfection, Expressing, Negative Control, Selection

Journal: PLoS ONE

Article Title: Considerations and practical implications of performing a phenotypic CRISPR/Cas survival screen

doi: 10.1371/journal.pone.0263262

Figure Lengend Snippet: (A) Adjusted volcano plot of screen data with low doxycycline (250ng/ml)/early harvest (24h after doxycycline removal, see ) showing the enrichment of sets of guides targeting genes not located on chromosome 5q or chromosome 19p. Blue points represent guide sets that are significantly enriched (P-value ≤ 0.01), Log2(fold change) ≥ 1), the green point is the positive control (DUX4), red points represent the non-target control guides. (B) Venn diagram showing the overlap of filtered hits between the four screens (EL: Early harvest-low Doxy, LL: Late harvest-Low doxy, EH: Early harvest-high Doxy, LH: Late harvest-High doxy), see also . (C) Viability staining showing surviving DIE cells containing single knockouts of potentials hits, identified in the CRISPR screen. Knockouts of individual genes were generated by transfection of sgRNA; 6 days later, cells were left untreated or treated with 3 different concentrations of doxycycline (100, 250 and 1000ng/ml) for and incubated for an additional 48–96 hours prior to visualizing surviving cells. Data are representative of at least three independent experiments. (D) Viability staining showing the surviving DIE-ieGFP-Cas9 cells (DIE cells expressing Cas9 constitutively and contain doxycycline-inducible eGFP) with single knockouts of mediator complex subunits. Knockouts of individual genes were generated by transfection of sgRNA; 6 days later cells were treated for doxycycline (250ng/ml) for 24h and incubated for an additional 48–96 hours prior to visualizing surviving cells. Data are representative of at least three independent experiments. (E) FACS data showing GFP-positive cells in surviving populations of DIE-ieGFP-Cas9 (expressing constitutive Cas9 and doxycycline-inducible GFP). cells containing single knockouts as indicated. Knockouts of individual genes were generated by transfection of sgRNA; 6 days later, cells were treated with doxycycline (250ng/ml) for 24h prior to FACS analysis. DIE-ieGFP-Cas9 cells comprised of 42% of eGFP-positive cells after DUX4 knockout. rtTA, MED25, MED24 and MED16 knockouts displayed a lower percentage of eGFP-expressing cells, comprising between 1.2–4% of eGFP-expressing cells. Data are representative of at least three independent experiments.

Article Snippet: The Human Brunello CRISPR knockout pooled lentiviral prep library was a gift from David Root and John Doench (Broad Institute, MA, U.S.A.).

Techniques: Positive Control, Control, Staining, CRISPR, Generated, Transfection, Incubation, Expressing, Knock-Out