Journal: eLife

Article Title: Genome-wide CRISPR screens of oral squamous cell carcinoma reveal fitness genes in the Hippo pathway

doi: 10.7554/eLife.57761

Figure Lengend Snippet: ( A ) Demographic details and genomic profile of the patients of which the 21 OSCC cell lines were derived from, with bar charts depicting the number of essential genes identified by MAGeCK. The presence of mutations/copy number alterations in the top five mutated genes in OSCC is shown. Numbers in first column indicated frequency of mutations (%) among OSCC tumors from TCGA while second column indicated frequency of mutation (%) among 21 OSCC cell lines. Bar charts in the lower panel shows the number of significant fitness genes (those with MAGeCK FDR less than or equal to 5%), with the orange bars representing the number of non-core fitness genes. Abbreviations: Ind – Indian; Chi – Chinese, Mal – Malay; Jap – Japanese, Cau – Caucasian; F – Female; M – Male; G – Gingiva; T – Tongue; BM – Buccal Mucosa; FOM – Floor of Mouth; LN – derived from lymph node metastasis. ( B ) Pie charts showing the proportion of fitness genes among the 18,010 genes screened. 918 non-core fitness genes were shortlisted after filtering out the core fitness genes. ( C ) Bar chart depicting the number of non-core fitness genes that are found in 1 to 21 dependent cell lines. Figure 1—source data 1. Analysis result from the genome-wide CRISPR-Cas9 screens.

Article Snippet: Recombinant DNA reagent , Human Improved Genome-wide Knockout CRISPR Library v1 (pooled library) , Addgene , #67989 , Containing 90,709 gRNAs, selectable with puromycin.

Techniques: Derivative Assay, Mutagenesis, Genome Wide, CRISPR

Journal: eLife

Article Title: Genome-wide CRISPR screens of oral squamous cell carcinoma reveal fitness genes in the Hippo pathway

doi: 10.7554/eLife.57761

Figure Lengend Snippet: ( A ) Schematic of the genome-wide CRISPR-Cas9 screening on 21 OSCC cell lines. ( B ) Workflow of CRISPR data processing and analysis pipeline, from raw sgRNA counts to the list of fitness genes that are significantly depleted during the genome-wide CRISPR screening and quantile normalized, batch corrected, scaled CRISPR score, using various bioinformatic tools/algorithms including CRISPRcleanR, MAGeCK and ComBat (indicated in Red font). For details, please refer to the Materials and methods section.

Article Snippet: Recombinant DNA reagent , Human Improved Genome-wide Knockout CRISPR Library v1 (pooled library) , Addgene , #67989 , Containing 90,709 gRNAs, selectable with puromycin.

Techniques: Genome Wide, CRISPR

Journal: eLife

Article Title: Genome-wide CRISPR screens of oral squamous cell carcinoma reveal fitness genes in the Hippo pathway

doi: 10.7554/eLife.57761

Figure Lengend Snippet: ( A ) Common oncogenic pathways altered among HNSCC samples of TCGA were annotated with frequency of dependency. Non-core fitness genes are indicated in red and the percentage of OSCC cell lines that were dependent on the genes are shown. The Drug Gene Interaction database (DGIdb) ( http://www.dgidb.org/ ) was used to determine if the gene is clinically actionable while the availability of drugs were determined using Open Targets Platform ( https://www.targetvalidation.org/ ). ( B ) Heatmap of gene essentiality of the 44 HNSCC cancer genes in the 21 OSCC lines. These are consensus cancer genes for HNSCC curated from and . ( C ) CRISPR scores of genes with driver mutations in at least one of the 21 OSCC cell lines. Cell lines labeled in green with mutation are examples of those showing oncogene addiction on mutated genes, for example on PIK3CA (ORL-150, BICR10, and HSC-2) and NFE2L2 (Ho-1-u-1). ( D ) Pathway enrichment analysis for fitness genes that are differentially enriched among the seven betel-quid associated OSCC. ( E ) Distribution of the 918 fitness genes based on their small molecule inhibitors tractability assessment. Tractability is defined as detailed in where: tractability group one included targets with approved drugs (Bucket 1) or drugs in clinical/pre-clinical development (Bucket 2, 3); tractability group two included targets with evidence supporting tractability albeit no drugs are available yet; while the least tractable group three included targets that lacks evidence informing tractability. Figure 2—source data 1. Analysis results of targetable genes and pathways in OSCC.

Article Snippet: Recombinant DNA reagent , Human Improved Genome-wide Knockout CRISPR Library v1 (pooled library) , Addgene , #67989 , Containing 90,709 gRNAs, selectable with puromycin.

Techniques: CRISPR, Labeling, Mutagenesis

Journal: eLife

Article Title: Genome-wide CRISPR screens of oral squamous cell carcinoma reveal fitness genes in the Hippo pathway

doi: 10.7554/eLife.57761

Figure Lengend Snippet: ( A ) KEGG pathway analysis of essential genes found on copy number amplified chromosome shows enrichment of pro-tumorigenic pathway including the Hippo signaling pathway. ( B ) The Hippo signaling pathway and annotation of genes that are OSCC fitness genes. ( C ) Oncoprint downloaded from cBioportal showing the copy number alterations of YAP1, WWTR1, PIK3CA, TP63, and SOX2 in the HNSCC samples (n = 295) (TCGA 1 ). Co-amplification of WWTR1 with PIK3CA can be seen in majority of the cases, whereby 40/59 (68%) tumors with PIK3CA amplification also have WWTR1 amplification. ( D ) Boxplots of CRISPR scores for the five genes, classified into two groups with (n = 11) or without (n = 10) copy number amplification of genes on chromosome 3q25-28. OSCC with copy number amplification showed significantly more negative CRISPR score for WWTR1, i.e. more dependent on WWTR1. This correlation was not found for PIK3CA. TP63 and SOX2 were not essential in any of the 21 OSCC cell lines. Whiskers of boxplot show minimum and maximum values, lines representing median. Unpaired two-tailed t-test was used for calculating statistical difference between two groups. ( E ) Baseline mRNA expressions of YAP1 and WWTR1 in selected OSCC cell lines were quantified by qPCR. YAP1-dependent ORL-48 and ORL-204 showed upregulation of YAP1 expression. While in other OSCC cell lines, the extend of WWTR1 overexpression is higher than YAP1. OKF6/htert1, an immortalized normal oral epithelial cell line is used as reference control for calculation of relative fold difference of gene expression. Data are shown as mean ± SD (n = 3 technical repeats). ( F ) Baseline protein expression of YAP1 and WWTR1 protein in selected OSCC cell lines. Actin was probed as a loading control.

Article Snippet: Recombinant DNA reagent , Human Improved Genome-wide Knockout CRISPR Library v1 (pooled library) , Addgene , #67989 , Containing 90,709 gRNAs, selectable with puromycin.

Techniques: Amplification, CRISPR, Two Tailed Test, Expressing, Over Expression, Control, Gene Expression

Journal: eLife

Article Title: Genome-wide CRISPR screens of oral squamous cell carcinoma reveal fitness genes in the Hippo pathway

doi: 10.7554/eLife.57761

Figure Lengend Snippet: ( A ) Essentiality profile (depicted with CRISPR scores heatmap) of YAP1, WWTR1, PIK3CA, TP63 and SOX2 across 21 OSCC cell lines derived from the CRISPR/Cas9 screen. Dependency on these genes were depicted as grey box in the bottom panel, according to the MAGeCK definition of significant depletion at FDR ≤ 0.05. No cell lines were dependent on TP63 or SOX2. The degree of essentiality differs across the lines. Some subsets of the cell lines are only dependent on either YAP1 or WWTR1, while neither gene appears to be essential in another subset of cell lines. PIK3CA, TP63 and SOX2 are genes implicated in HNSCC carcinogenesis that are often co-amplified with WWTR1, located on chromosome 3q25-28. All WWTR1-dependent cell lines had copy number amplification on these genes while all PIK3CA mutated cell lines are not dependent on either YAP1 or WWTR1. ( B ) Western blot images showing the protein level of YAP1 and WWTR1 on day 4 upon transducing the Cas-9 expressing cell lines with lentivirus carrying gene-specific sgRNA. Two sgRNAs were used per target gene. ( C ) Co-competition assay was used to validate the essentiality of YAP1 and WWTR1 on the selected cell lines. The growth of the BFP-positive transduced population was compared to the non-transduced population throughout the 18 days assay. The percentage of BFP-positive cells obtained at different time points were normalized to the day 4 readings for respective sgRNA (except ORL-204 which had time points normalized to the day 6 readings for respective sgRNA). PLK1 is a core essential gene included as a positive control. Negative controls include CHAT which is a non-essential gene across the panel of cell lines, and NT serves as a non-targeting control. Data are shown as mean ± SD (n = 2 biological repeats). ( D ) qPCR results show suppression of downstream targets of YAP1 and WWTR1 only when the respective fitness gene is being knocked-out. Down-regulation of CTGF and CYR61 gene expression was observed when YAP1 is knocked-out in the YAP1-dependent cell lines (ORL-48 and ORL-204). In the WWTR1-dependent cell lines (ORL-214, PE/CA-PJ15), CTGF and CYR61 expression is only suppressed when WWTR1 is knocked-out. Data are shown as mean ± SD (n = 2 independent experiments with technical triplicates). Figure 3—source data 1. All raw data related to and its figure supplements on analysis result of YAP1 and WWTR1 as fitness genes for OSCC.

Article Snippet: Recombinant DNA reagent , Human Improved Genome-wide Knockout CRISPR Library v1 (pooled library) , Addgene , #67989 , Containing 90,709 gRNAs, selectable with puromycin.

Techniques: CRISPR, Derivative Assay, Amplification, Western Blot, Expressing, Competitive Binding Assay, Positive Control, Control, Gene Expression

Journal: eLife

Article Title: Genome-wide CRISPR screens of oral squamous cell carcinoma reveal fitness genes in the Hippo pathway

doi: 10.7554/eLife.57761

Figure Lengend Snippet: ( A ) Plot of differential essentiality between YAP1 and WWTR1 reveal correlation of essentiality with gene expression. Gene expression and CRISPR score of YAP1 and WWTR1 from Project Score on 273 cancer cell lines were extracted. Those lines that are more dependent on YAP1 over WWTR1 (negative differential CRISPR score) are mostly with high YAP1 (blue) and low WWTR1 (red) expression; On the other hand, cell lines that are more dependent on WWTR1 are among those with overexpression of WWTR1 (red). ( B ) Significant negative correlation between WWTR1 gene expression and gene essentiality was evident in the 273 cancer cell lines screened in Project Score. Those cell lines that are dependent on WWTR1 (negative CRISPR score) tend to have higher WWTR1 expression. ( C ) WWTR1 gene expression and gene essentiality also showed non-significant negative correlation among the 21 OSCC lines screened. ( D ) Other cancer types showing significant negative correlation between WWTR1 gene expression and gene essentiality include non-small cell lung carcinoma, squamous cell lung carcinoma, glioblastoma, breast carcinoma, and lung adenocarcinoma (data from Project Score).

Article Snippet: Recombinant DNA reagent , Human Improved Genome-wide Knockout CRISPR Library v1 (pooled library) , Addgene , #67989 , Containing 90,709 gRNAs, selectable with puromycin.

Techniques: Gene Expression, CRISPR, Expressing, Over Expression

Journal: eLife

Article Title: Genome-wide CRISPR screens of oral squamous cell carcinoma reveal fitness genes in the Hippo pathway

doi: 10.7554/eLife.57761

Figure Lengend Snippet: ( A ) Clonogenic assay showing inhibitory effect upon knocking out respective fitness genes in YAP1- and WWTR1-dependent lines. In BICR10 and HSC-2 knocking out of either YAP1 or WWTR1 with sgRNAs did not have any impact on clonogenicity. Experiment is performed in technical triplicate and repeated twice. Representative image of one technical repeat is shown. ( B ) sgRNA level log fold change from triplicate CRISPR screens of SAS. Depletion were seen only in sgRNAs targeting exon 1–4 of YAP1, which were present in YAP1-MAML2 fusion protein, but not seen in sgRNA 6–6 which targets exon 6. Data are shown as mean ± SD (n = 3 biological repeats). ( C ) Western blot to check efficacy of target protein knockout using CRISPR/Cas9 on SAS. Y1K is one of the sgRNA from the KY library v1, targeting exon 4, while Y2B is an independent sgRNA targeting exon 1 of YAP1. ( D ) Co-competition assay results for SAS, validating its dependency on YAP1. Previous work by 2 revealed oncogenic fusion of YAP1 with MAML2 in SAS, rendering the cells to be dependent on the fusion protein. Both our genome-wide screening data and validation data have also confirmed the preferential dependency of SAS on YAP1, over its paralog, WWTR1. ( E ) Clonogenicity assay confirmed preferential dependency of SAS on YAP1. ( F ) qPCR of downstream target genes showed relatively stronger suppression when the fitness gene, YAP1 is depleted with sgRNA Y1K or Y2B. Data are shown as mean ± SD (n = 2 independent experiments with technical triplicates).

Article Snippet: Recombinant DNA reagent , Human Improved Genome-wide Knockout CRISPR Library v1 (pooled library) , Addgene , #67989 , Containing 90,709 gRNAs, selectable with puromycin.

Techniques: Clonogenic Assay, CRISPR, Western Blot, Knock-Out, Competitive Binding Assay, Genome Wide, Biomarker Discovery

Journal: eLife

Article Title: Genome-wide CRISPR screens of oral squamous cell carcinoma reveal fitness genes in the Hippo pathway

doi: 10.7554/eLife.57761

Figure Lengend Snippet:

Article Snippet: Recombinant DNA reagent , Human Improved Genome-wide Knockout CRISPR Library v1 (pooled library) , Addgene , #67989 , Containing 90,709 gRNAs, selectable with puromycin.

Techniques: Recombinant, Genome Wide, Knock-Out, CRISPR, Plasmid Preparation, Cloning, Fluorescence, Transfection, Derivative Assay, Western Blot, Purification, Reverse Transcription, SYBR Green Assay, Software

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: The FASEB Journal

Article Title: Identification of calcium and integrin‐binding protein 1 as a novel regulator of production of amyloid β peptide using CRISPR/Cas9‐based screening system

doi: 10.1096/fj.201902966rr

Figure Lengend Snippet: FIGURE 3 Disruption of Cib1 increases Aβ level without affecting Aβ production-related protein expressions. A, Immunoblotting in Cib1- KO monoclonal cell lines using antibodies against CIB1 and α-tubulin. Two Cib1-KO monoclonal cell lines were generated by CRISPR/Cas9 system. B, The relative secreted Aβ40 and Aβ42 level in (A). Secreted Aβ were measured by two-site ELISA (n = 4, mean ± SEM, P values were assessed by one-way ANOVA with Dunnett's HSD post hoc analysis). C, The relative secreted Aβ40 and Aβ42 level. Mouse CIB1 was expressed in Cib1-KO monoclonal cell (n = 3, mean ± SEM, P values were assessed by one-way ANOVA with Tukey's HSD post hoc analysis). D, Immunoblot analysis in (C). The expression of CIB1, α-tubulin, and Aβ-production associated proteins, including APP, BACE1, sAPPβ in cultured media, Nct, PS1, and PS2, were analyzed by western blotting. mNct: mature Nct, imNct: immature Nct, PS1C: C-terminal fragment of PS1, PS1N: N-terminal fragment of PS1, PS2C: C-terminal fragment of PS2, PS2N: N-terminal fragment of PS2

Article Snippet: For gRNA packaging, 50947 was modified as previously described.21 For the production of gRNA lentivirus for interesting genes, the gRNA sequences (Table 1) were inserted into the lentiviral vector (Addgene #50947). cDNAs encoding mouse WT CIB1 was cloned into pcDNA3.1(+)-Hygromycin (Addgene #V87020) by Gibson Assembly (Master Mix, New England BioLabs, Ipswich, MA, USA).

Techniques: Disruption, Western Blot, Generated, CRISPR, Enzyme-linked Immunosorbent Assay, Expressing, Cell Culture

Journal: The FASEB Journal

Article Title: Identification of calcium and integrin‐binding protein 1 as a novel regulator of production of amyloid β peptide using CRISPR/Cas9‐based screening system

doi: 10.1096/fj.201902966rr

Figure Lengend Snippet: FIGURE 4 CIB1 interacts with active γ-secretase in the DKO cell. Representative immunoblot for immunoprecipitation with anti-PS2 antibody. The active γ-secretase and CIB1 were co- immunoprecipitated (IP) with anti-C-terminally PS2 rabbit antibody in lysates of PS1-/PS2-double knockout MEF (DKO) and PS2 overexpressing DKO cells. Immunoprecipitated proteins were analyzed by Western blotting with antibodies against N- and C-terminal fragments of PS2, Nct, Pen-2, and CIB1. Lysate precipitated without antibody served as a negative control. mNct: mature Nct, imNct: immature Nct, PS2C: C-terminal fragment of PS2, PS2N: N-terminal fragment of PS2

Article Snippet: For gRNA packaging, 50947 was modified as previously described.21 For the production of gRNA lentivirus for interesting genes, the gRNA sequences (Table 1) were inserted into the lentiviral vector (Addgene #50947). cDNAs encoding mouse WT CIB1 was cloned into pcDNA3.1(+)-Hygromycin (Addgene #V87020) by Gibson Assembly (Master Mix, New England BioLabs, Ipswich, MA, USA).

Techniques: Western Blot, Immunoprecipitation, Double Knockout, Negative Control

Journal: The FASEB Journal

Article Title: Identification of calcium and integrin‐binding protein 1 as a novel regulator of production of amyloid β peptide using CRISPR/Cas9‐based screening system

doi: 10.1096/fj.201902966rr

Figure Lengend Snippet: FIGURE 5 Downregulation of CIB1 does not affect the intrinsic enzymatic activity of γ-secretase. A, Immunoblotting of CIB1 in the Cib1-siRNA treated N2a cell. B, De novo Aβ generation in in vitro γ-secretase assay using membrane fraction from (A). PS1-/PS2-double knockout MEF (DKO) cell with PS1 or PS2 overexpression served as a positive control. Generated Aβ were measured by two-site ELISA (n = 9, mean ± SEM, P values of right 4 columns were assessed by one-way ANOVA with Dunnett's HSD post hoc analysis). C, Immunoblotting of CIB1 in Cib1-KO N2a clone. D, De novo Aβ generation in in vitro γ-secretase assay using membrane fraction from (C). As described in (B), DKO cell with PS1 or PS2 overexpression served as a positive control. Generated Aβ were measured by two-site ELISA (n = 9, mean ± SEM, P values of right 2 columns were assessed by Student's t test)

Article Snippet: For gRNA packaging, 50947 was modified as previously described.21 For the production of gRNA lentivirus for interesting genes, the gRNA sequences (Table 1) were inserted into the lentiviral vector (Addgene #50947). cDNAs encoding mouse WT CIB1 was cloned into pcDNA3.1(+)-Hygromycin (Addgene #V87020) by Gibson Assembly (Master Mix, New England BioLabs, Ipswich, MA, USA).

Techniques: Activity Assay, Western Blot, In Vitro, Membrane, Double Knockout, Over Expression, Positive Control, Generated, Enzyme-linked Immunosorbent Assay

Journal: The FASEB Journal

Article Title: Identification of calcium and integrin‐binding protein 1 as a novel regulator of production of amyloid β peptide using CRISPR/Cas9‐based screening system

doi: 10.1096/fj.201902966rr

Figure Lengend Snippet: FIGURE 6 Depletion of CIB1 decreases the cell surface localization of mature Nicastrin. Cell surface biotinylation assay in Cib1-knockdown (A,B) or Cib1- KO (clone #2) N2a cells (C,D). A and C, Representative immunoblotting of cell surface biotinylation for Nicastrin. Nicastrin expressed on the cell surface were labeled by NHS-SS-Biotin, and selectively pulled down by streptavidin beads. mNct: mature Nct, imNct: immature Nct. B, Quantification of band intensities of mNct and cell surface mNct in (A) (n = 4, mean ± SEM, P values of right 3 columns were assessed by one- way ANOVA with Dunnett's HSD post hoc analysis). D, Quantification of band intensities of mNct and cell surface mNct in (C) (n = 4, mean ± SEM, P values were assessed by Student's t test)

Article Snippet: For gRNA packaging, 50947 was modified as previously described.21 For the production of gRNA lentivirus for interesting genes, the gRNA sequences (Table 1) were inserted into the lentiviral vector (Addgene #50947). cDNAs encoding mouse WT CIB1 was cloned into pcDNA3.1(+)-Hygromycin (Addgene #V87020) by Gibson Assembly (Master Mix, New England BioLabs, Ipswich, MA, USA).

Techniques: Cell Surface Biotinylation Assay, Knockdown, Western Blot, Labeling

Journal: The FASEB Journal

Article Title: Identification of calcium and integrin‐binding protein 1 as a novel regulator of production of amyloid β peptide using CRISPR/Cas9‐based screening system

doi: 10.1096/fj.201902966rr

Figure Lengend Snippet: FIGURE 7 Scheme of mechanism on regulation of Aβ production by CIB1. We demonstrated in the present study that CIB1 maintains the expression of the γ-secretase on the cell surface (left panel). On the contrary, CIB1 downregulation facilitates the internalization of the γ-secretase (right panel), which leads to the increased Aβ production. The line with arrows showed the subcellular trafficking of the γ-secretase. The dotted line indicates the reduced transport of the γ-secretase

Article Snippet: For gRNA packaging, 50947 was modified as previously described.21 For the production of gRNA lentivirus for interesting genes, the gRNA sequences (Table 1) were inserted into the lentiviral vector (Addgene #50947). cDNAs encoding mouse WT CIB1 was cloned into pcDNA3.1(+)-Hygromycin (Addgene #V87020) by Gibson Assembly (Master Mix, New England BioLabs, Ipswich, MA, USA).

Techniques: Expressing

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