Journal: BMC Biology

Article Title: Genome-scale CRISPR screening at high sensitivity with an empirically designed sgRNA library

doi: 10.1186/s12915-020-00905-1

Figure Lengend Snippet: Selected Cas9-expressing single cell clones show stronger editing efficiency compared to a Cas9 bulk population. a Workflow for the selection of Cas9 single-cell clones (SCCs). SCCs were sorted from the HAP1 Cas9 bulk population and further characterized. Cas9 editing was assessed by cell surface marker knockout followed by FACS staining and cell viability upon knockout of a core essential gene. Two highly editing single-cell clones (SCC11 and SCC12) were selected for further experiments. b HAP1 Cas9 bulk, Cas9 SCC11, and Cas9 SCC12 cells were transfected with the HDCRISPRv1 vector encoding an sgRNA targeting either the safe harbor locus AAVS1 as a control or the core essential gene RNA Polymerase 2 subunit E ( POLR2E ). Editing efficiency based on cell viability of sgPOLR2E-transfected cells in comparison to sgAAVS1 control cells was addressed by crystal violet staining. The number of surviving cells was strongly reduced in cells transfected with an sgRNA directed against POLR2E ( n = 3 for each cell line and sgRNA). c Editing efficiency was furthermore assessed upon transduction of HAP1 Cas9 bulk, Cas9 SCC11, and Cas9 SCC12 cells with the HDCRISPRv1 vector expressing sgRNAs targeting the surface marker CD46 , followed by FACS staining of residual CD46 protein to address knockout efficiency. Antibody staining of the non-edited cell lines was used as a control. Lines represent the mean of independent measurements ( n = 3 for each cell line and condition)

Article Snippet: The HAP1 cell line was authenticated using Multiplex Cell Authentication by Multiplexion (Heidelberg, Germany) as described recently [ ].

Techniques: Expressing, Clone Assay, Selection, Marker, Knock-Out, Staining, Transfection, Plasmid Preparation, Control, Comparison, Transduction

Journal: BMC Biology

Article Title: Genome-scale CRISPR screening at high sensitivity with an empirically designed sgRNA library

doi: 10.1186/s12915-020-00905-1

Figure Lengend Snippet: The HD CRISPR library efficiently identifies core, non- and context-dependent essential genes. a Workflow of a pilot screen conducted with the HD CRISPR library in HAP1 cells. The screen was performed in parallel in the Cas9-expressing bulk population and two highly editing single cell clones for both libraries independently. Successfully transduced cells were selected with puromycin for 48 h and then split into two independent replicates. The screen was performed for a duration of 14 days. b Core essential genes were strongly depleted over the course of screening with either of the two libraries, HD CRISPR libraries A and B, in contrast to nonessential genes. Stronger depletion was observed in the two single cell clones with high Cas9 editing efficiency. c Empirical cumulative distribution function for viability screens conducted with different HAP1 Cas9 cell lines and both HD CRISPR sub-libraries. Shown are results for sgRNAs targeting genes from core essential or nonessential gene sets or representing non-targeting controls. d Area under the curve (AUC) values for individual replicates of the empirical cumulative distribution functions shown in c . e Comparison of HAP1 essential genes as identified with the HD CRISPR library, a gene trap screen by Blomen et al. and two CRISPR screens using either the TKOv1 or TKOv3 library by Hart et al. . f Number of essential genes detected with increasing number of sgRNAs per gene using BAGEL (left; BF > 6) or MAGeCK RRA (right; FDR < 5%). sgRNAs were subsampled from the combined HD CRISPR library (sub-libraries A and B). Each data point represents the average of 5 samples. Error bars are ±1 s.e.m

Article Snippet: The HAP1 cell line was authenticated using Multiplex Cell Authentication by Multiplexion (Heidelberg, Germany) as described recently [ ].

Techniques: CRISPR, Expressing, Clone Assay, Comparison

Journal: BMC Biology

Article Title: Genome-scale CRISPR screening at high sensitivity with an empirically designed sgRNA library

doi: 10.1186/s12915-020-00905-1

Figure Lengend Snippet: CRISPR screens conducted at a high dynamic range predict the cutting efficiency of sgRNAs based on mild viability phenotypes. a A mixture model was used to divide control sgRNAs of the HD CRISPR library A in the single cell clone SCC12 screen into three groups: (1) sgRNAs with a target-dependent viability phenotype (red), (2) sgRNAs with a small target-independent phenotype likely caused by a double-strand break (blue), and (3) sgRNAs with no phenotype due to a lack of DNA cutting (yellow). Log2 fold change distributions of targeting and non-targeting control sgRNAs are indicated as dashed and solid curves, respectively. b Number of sgRNAs targeting nonessential HAP1 genes associated with each phenotype group. Nonessential genes were determined using MAGeCK which requires no prior knowledge for analysis. sgRNAs are stratified based on their design: “empirical essential” sgRNAs target context-specific essential genes and were selected for the HD CRISPR library based on their previous on-target phenotypes. “Empirical nonessential” sgRNAs are part of previously published libraries and target broadly nonessential genes. They were selected based on their lack of outlier phenotypes. De novo sgRNAs were designed using the software cld

Article Snippet: The HAP1 cell line was authenticated using Multiplex Cell Authentication by Multiplexion (Heidelberg, Germany) as described recently [ ].

Techniques: CRISPR, Control, Software

Journal: BMC Biology

Article Title: Genome-scale CRISPR screening at high sensitivity with an empirically designed sgRNA library

doi: 10.1186/s12915-020-00905-1

Figure Lengend Snippet: HAP1 cells are highly dependent on Yamanaka factors and the Fanconi anemia pathway. a HAP1 context-dependent essential genes are enriched for genes comprising the Fanconi anemia pathway as well as factors known to induce pluripotency. The y -axis provides an estimate of the percentage of essentiality in other cell lines previously screened. Fanconi anemia pathway genes are highlighted in red, Yamanaka factors are shown in blue. HAP1-specific nonessential genes are highlighted in green. nt ctrl = non-targeting control. b Crystal violet staining of HAP1 cells treated with various siRNAs targeting SOX2 , POU5F1 , and KLF4 and a non-targeting control. Shown is a representative result of three independent experiments. c Essentiality of known cancer drivers, Fanconi anemia pathway genes, and pluripotency factors across cell lines representing different cancer cell types in comparison to HAP1 cells and human embryonic stem cells

Article Snippet: The HAP1 cell line was authenticated using Multiplex Cell Authentication by Multiplexion (Heidelberg, Germany) as described recently [ ].

Techniques: Control, Staining, Comparison