Journal: Genome Research

Article Title: Genome-scale identification of cellular pathways required for cell surface recognition

doi: 10.1101/gr.231183.117

Figure Lengend Snippet: A cell-based genome-scale CRISPR-KO approach identifies pathways required for monoclonal antibody surface epitope recognition. ( A ) Schematic of the approach based on CRISPR/Cas9 technology using a genome-scale lentiviral gRNA library. Genes identified as being required for surface display of the epitope recognized by an anti-CD58 ( B ) and anti-GYPA ( C ) mAbs. The enrichment of gRNAs targeting each gene is quantified as the robust rank aggregation (RRA) score calculated using the MAGeCK software between selected cells that had lost the mAb epitope versus control cells and is shown plotted in rank order. ( D ) Genes identified as being required for surface display of the epitope recognized by an anti-CD59 mAb; here, genes are ordered alphabetically for clarity. Circles represent individual genes and are sized according to their false-discovery rate (FDR): large circle = FDR < 1%, small circle = 1% < FDR < 5%. Genes encoding the direct receptors are indicated with gray circles. Only genes with FDR < 5% are named and are color-coded according to their function.

Article Snippet: The Human Improved Genome-Wide Knockout CRISPR Library v1 (Addgene no. 67989), lentiviral Cas9 reporter plasmids pKLV2-U6gRNA5(gGFP)-PGKBFP2AGFP-W (Addgene no. 67980) and pKLV2-U6gRNA5(Empty)-PGKBFP2AGFP-W (Addgene no. 67979), lentiviral vector expressing Cas9 fused with the blasticidin-resistant gene at the C terminus pKLV2-EF1a-Cas9Bsd-W (Addgene no. 68343), and lentiviral CRISPR gRNA expression vector pKLV2-U6gRNA5(BbsI)-PGKpuro2ABFP-W (Addgene no. 67974) were used in this study.

Techniques: CRISPR, Software, Control

Journal: PLoS ONE

Article Title: CRISPR/Cas-based customization of pooled CRISPR libraries

doi: 10.1371/journal.pone.0199473

Figure Lengend Snippet: The gRNA-encoding plasmid DNA in the pooled CRISPR library contained the nucleotide sequence 5’-CGG-3’, which could be used as the PAM sequence for Cas9 proteins. Cas9 RNPs, which are complexes of Cas9 proteins and rc-gRNAs, could selectively cleave plasmid DNA possessing complementary sequences with rc-gRNA. Based on this principle, only the desired gRNAs could be depleted from the pooled CRISPR library.

Article Snippet: For generating the gRNA-depleted library, 20 μg of pooled CRISPR library plasmids DNA (human GeCKO v1 and v2 lentiviral sgRNA libraries, Addgene #1000000049) was incubated with NEBuffer 3.1 containing 1× Cas9 RNPs and a complex of 10 μg of Cas9 proteins and 7.5 μg of rc-gRNAs for 8 h at 37°C.

Techniques: Plasmid Preparation, CRISPR, Sequencing

Journal: Nucleic acids research

Article Title: Uridine-cytidine kinase 2 potentiates the mutagenic influence of the antiviral β-d-N4-hydroxycytidine.

doi: 10.1093/nar/gkad1002

Figure Lengend Snippet: Figure 1. Identification of genetic factors that modify response to EIDD-1931. ( A ) Schematic of the CRISPR / Cas9 screens to identify genes that modulate EIDD-1931 response. ( B ) Gene le v el summary of guide enrichment in two E μ-Myc lymphoma cell lines (AH15A and AF47A) selected with 5 μM EIDD-1931 o v er 4 rounds. The representation of the guides (4–5 per gene) was compared to baseline (day 3) and shown on a plot of -Log10 False Disco v ery Rate (FDR). Dotted lines mark an FDR value of 0.05. Uck2 (in red) had the lo w est FDR v alues in both lines. Impact of Uck2 o v ere xpression and loss on the sensitivity to EIDD-1931 in E μ-Myc lymphoma cell lines ( C ) and HoxA9-Meis1 myeloid cell lines ( D ). ( E ) Immunoblots for Uck2 in Uck2 -OE, WT, Uck2 + / −and Uck2 −/ −E μ-Myc lymphoma cell lines. HSP70 serves as a loading control. CellTiter-Glo assays were used to assess the impact of homozygous loss of Uck1 ( F ) or heterozygous loss of Cmpk1 ( G ) on the sensitivity of E μ-Myc lymphoma cell lines to EIDD-1931. ( H ) Impact of Uck2 on the sensitivity of E μ-Myc lymphoma cell lines to azacitidine. ( I ) Overview of ribonucleoside processing and contribution to RNA and DNA synthesis. ( J ) γ-H2AX staining analysis of Uck2 over-expressing, parental E μ-Myc lymphoma cells and isogenic Uck2 −/ −derivatives treated with 1 μM EIDD-1931 for 24 h. Parental cells treated with 1 μM Cisplatin for 24 h were used as a control. The positive cell fraction was determined based on gating on the untreated parental control. Data shown in C, D, F, G, H and J are means ± 1 SD from multiple replicate wells across 3–5 independent experiments, taken at 48 h (C,D,F and G) or 24h (H and J). Three independent CRISPR / Cas9 edited clones were used for Uck2 −/ −(C and D) and Cmpk1 + / −(G), whereas two independent clones were used for other genotypes in C, D, F and H. Some parental (WT) control data are shared between C and F.

Article Snippet: Genome-wide CRISPR / Cas9 knockout screen The Genome-wide Mouse Lentiviral CRISPR gRNA Library v1 was used for our screen ( Addgene, Pooled Library #50947 ) ( 18 ) .

Techniques: CRISPR, Western Blot, Control, DNA Synthesis, Staining, Expressing, Clone Assay

Journal: bioRxiv

Article Title: Functional single-cell genomics of human cytomegalovirus infection

doi: 10.1101/775080

Figure Lengend Snippet: Host-directed CRISPR screens identify host dependency and restriction factors. ( A ) Experimental design for pooled, host-directed CRISPRi/n screening. Genome-wide sgRNA libraries targeting human genes with multiple sgRNAs each were lentivirally delivered into primary human foreskin fibroblasts expressing the CRISPRi/n machineries, followed by infection with HCMV. sgRNA cassettes were quantified by deep sequencing in the initial (t 0 ) population, the surviving population and an uninfected control population to account for gene essentiality in absence of infection. ( B ) Results of the host-directed CRISPRi screen displayed as a scatter plot of average gene essentiality (i.e. infection-independent phenotype; y-axis) vs. protection/sensitization to death upon HCMV infection (i.e. infection-dependent phenotype; x-axis).

Article Snippet: For targeting host genes, we used the human CRISPRi v2 library (Addgene #83969) , and the K. Yusa et al. human knockout CRISPR v1 library (Addgene #67989) , respectively.

Techniques: CRISPR, Genome Wide, Expressing, Infection, Sequencing

Journal: bioRxiv

Article Title: Functional single-cell genomics of human cytomegalovirus infection

doi: 10.1101/775080

Figure Lengend Snippet: ( A ) Results of host-directed CRISPRn screen displayed as a scatter plot of average gene essentiality (i.e. infection-independent phenotype; y-axis) vs. protection/sensitization to death upon HCMV infection (i.e. infection-dependent phenotypes; x-axis). Note that due to the experimental design of the screen, the apparent gene essentiality phenotypes are underestimating the real essentiality because t 0 refers to the beginning of HCMV infection, not lentiviral delivery of the sgRNA library. ( B ) Direct comparison of CRISPRi and CRISPRn phenotypes for host targets represented in both libraries. Hits involved in viral adhesion and entry, as well as host cell survival or apoptosis are more pronounced in the CRISPRn screen. Cullin/RING pathway members and some vesicle trafficking factors were only resolved in the CRISPRi screen.

Article Snippet: For targeting host genes, we used the human CRISPRi v2 library (Addgene #83969) , and the K. Yusa et al. human knockout CRISPR v1 library (Addgene #67989) , respectively.

Techniques: Infection

Journal: bioRxiv

Article Title: Functional single-cell genomics of human cytomegalovirus infection

doi: 10.1101/775080

Figure Lengend Snippet: ( A ) Numbers of single cells for each sgRNA target for each experimental time point in the host-directed CRISPRi Perturb-seq experiment. The average is 165 ± 50 (mean ± standard deviation) cells per sgRNA per time point. ( B ) Knockdown levels for each sgRNA target calculated from the expression of the target gene in cells with a given sgRNA target relative to cells with control sgRNAs. No transcript at all was detected for VTCN1. Median knockdown level was 87.1 %. ( C ) Hierarchical clustering of expression changes of the most variable 100 genes (excluding the targeted factors) in response to host factor knockdown in naïve cells, relative to naïve cells with control sgRNAs. ( D, E, F, G ) UMAP projections of single-cell transcriptomes of cells from the host-directed Perturb-seq experiment (same as in ), color-coded by experimental time post infection (D), percentage of viral transcripts per cell (E), interferon score, calculated from the normalized expression of interferon stimulated genes (F), and by pathway of the targeted host factor in each cell (G). ( H ) Cluster membership as a function of sgRNA target and time post infection.

Article Snippet: For targeting host genes, we used the human CRISPRi v2 library (Addgene #83969) , and the K. Yusa et al. human knockout CRISPR v1 library (Addgene #67989) , respectively.

Techniques: Standard Deviation, Expressing, Infection

Journal: bioRxiv

Article Title: Functional single-cell genomics of human cytomegalovirus infection

doi: 10.1101/775080

Figure Lengend Snippet: ( A ) Numbers of single cells for each sgRNA target for each experimental time point in the host-directed CRISPRi Perturb-seq experiment. The average is 188 ± 77 (mean ± standard deviation) cells per sgRNA per time point. Note the over-proportional drop in numbers in late time points of cells with apoptosis-related sgRNA targets. ‘Control’ denotes all safe-targeting sgRNAs, which are 4 and 5 distinct sgRNAs targeting the host and virus, respectively. ( B ) Violin plots of the distribution of viral RNA fraction per cell as a function of time post infection and the sgRNA target (red, protective phenotype; blue, sensitizing phenotype; grey, control). Regions of the violin plot corresponding to uninfected cells, as well as early and late stages of infection are highlighted. Note that uninfected cells have non-zero background amounts of viral RNA, and those background levels are higher in later time points, indicating leaking of viral RNA from dying cells. ( C, D ) Cluster membership as a function of sgRNA target and time post infection for cells with host-targeting sgRNAS (C) and virus-targeting sgRNAs (D).

Article Snippet: For targeting host genes, we used the human CRISPRi v2 library (Addgene #83969) , and the K. Yusa et al. human knockout CRISPR v1 library (Addgene #67989) , respectively.

Techniques: Standard Deviation, Infection