Journal: Nature Communications

Article Title: A pan-CRISPR analysis of mammalian cell specificity identifies ultra-compact sgRNA subsets for genome-scale experiments

doi: 10.1038/s41467-022-28045-w

Figure Lengend Snippet: a Schematic illustrating difference between classic synthetic lethality and our common genetic architecture . Synthetic lethality consists of many individual Y i functions. These functions are cell-type-specific models with single features. Our proposed common genetic architecture is hypothesized to connect these “private” functions with shared CERES features. A common genetic architecture has many redundant edges, and more interconnected nodes. More nodes suggest that more cell-type-specific phenotypes are predictable, and more edges suggest redundancy. b A network built from the aggregation of all multivariate models. Genes are represented as nodes and feature-target gene relations as edges. Colors represent distinct subnetwork communities that were identified by the Louvain method. c Network communities with (right) and without (left) nodes/edges involving functional CRISPR features for a single Louvain community, and a comparison with our hypothesis from ( a ). Edges are colored based upon the data source; and nodes are colored based on the model score (of top ten feature model) of the corresponding gene as target. d To quantitate the visual similarity between our hypothesis in ( a ) and the data in ( c ) across all Louvain communities, we examined the differences in the clustering coefficient, the average number of neighbors, and the network heterogeneity. e gprofiler2 plots examine the enrichment of functional categories. f Residual plot identifies GO terms that are more (residuals of −log10 P values >10) or less (residuals of -log10 P values < −10) enriched in predictor genes than in target genes. Dots represent shared GO terms among the 100 most significant terms in target and predictor gprofiler2 analysis result. The p- values from gprofiler2 for ( e ) and ( f ) are based on hypergeometric tests with multiple testing corrections using the g:SCS method. Source data are provided as a Source Data file.

Article Snippet: Human Brunello CRISPR knockout pooled library was a gift from David Root and John Doench (Addgene #73178).

Techniques: Functional Assay, CRISPR, Comparison

Journal: Nature Communications

Article Title: A pan-CRISPR analysis of mammalian cell specificity identifies ultra-compact sgRNA subsets for genome-scale experiments

doi: 10.1038/s41467-022-28045-w

Figure Lengend Snippet: a Two separate pooled screens were performed in a cell line (PC9) that was not included in model training and validation. Experiment 1 was the full Brunello library. A 21-day dropout experiment was performed in PC9 cells. Measurements on 18,114 genes were direct and form the gold standard. The L200 can be computationally extracted from the full screen and compared to these gold-standard measurements. A new L200 standalone library of 800 guides targeting 200 genes was cloned. This library can be used to perform a small-scale lossy compression experiment. The data can then be compared to the gold standard. b Correlations of inferred vs measured CERES scores for both screens in ( a ) and a comparison of the predictions between the standalone sets and the computationally extracted L200 set in the Brunello library. c A Venn diagram describes the overlap in “Hits” in the 500 most differentially required genes for growth in PC9 cells. Both of the lossy compression screens from ( a ) and the gold-standard (measured) data are compared. Source data are provided as a Source Data file.

Article Snippet: Human Brunello CRISPR knockout pooled library was a gift from David Root and John Doench (Addgene #73178).

Techniques: Biomarker Discovery, Clone Assay, Comparison

Journal: Hepatology (Baltimore, Md.)

Article Title: Depletion of TRRAP Induces p53-Independent Senescence in Liver Cancer by Down-Regulating Mitotic Genes.

doi: 10.1002/hep.30807

Figure Lengend Snippet: Figure 1. A kinome CRISPR screen identifies TRRAP as an essential gene for HCC cell growth. A) Three HCC cell lines were used to identify genes whose depletion inhibit HCC proliferation. Eight candidate genes were identified by integrating CRISPR screen results with HCC patient data. B) Genes from the CRISPR screen were ranked by their false discovery rate, the 8 candidate genes are indicated. C) mRNA levels of TRRAP in non- tumor and tumor samples from the GSE14520 (left) and TCGA (right) data sets, p-values were calculated using moderated t-test and Wilcoxon signed-rank test respectively. Black lines indicate the geometric mean of each group. D) Kaplan Meier curves of HCC patients from the TCGA (left) and NIH Laboratory of Experiment Carcinogenesis (LEC, right) cohorts with high (TCGA n=115 and LEC n=31) or low (TCGA n=118 and LEC n=31) TRRAP expression. P-values were calculated using the log-rank Mantel-Cox test. *p < 0.05, ***p < 0.001.

Article Snippet: Kinome CRISPR screen The human kinome CRISPR pooled library was a gift from John Doench and David Root (Addgene #1000000083).

Techniques: CRISPR, Expressing

Journal: Hepatology (Baltimore, Md.)

Article Title: Depletion of TRRAP Induces p53-Independent Senescence in Liver Cancer by Down-Regulating Mitotic Genes.

doi: 10.1002/hep.30807

Figure Lengend Snippet: Figure 4. Senescence induced by TRRAP and KAT5 depletion is independent of p21. A) Schematic illustrating generation of p21 and TRRAP/KAT5 double knockout cells using CRISPR. B) Western blot analysis of TRRAP, KAT5 and p21 levels in Huh7 and SNU-475 cells infected with the indicated sgRNAs. C) Colony formation and SA-β-gal staining of Huh7 cells infected with the indicated sgRNAs. NS = not significant (student’s t test).

Article Snippet: Kinome CRISPR screen The human kinome CRISPR pooled library was a gift from John Doench and David Root (Addgene #1000000083).

Techniques: Double Knockout, CRISPR, Western Blot, Infection, Staining

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