human improved genome-wide knockout crispr library v1 addgene #67989  (Addgene inc)

Journal: Scientific reports

Article Title: CRISPR-Cas9 genetic screens reveal regulation of TMPRSS2 by the Elongin BC-VHL complex.

doi: 10.1038/s41598-025-95644-0

Figure Lengend Snippet: Fig. 3. Genome-wide and targeted CRISPR-Cas9 screens reveal Elongin B and VHL as regulators of TMPRSS2 expression. (A) Schematic workflow of the genome-wide and targeted CRISPR-Cas9 screens. Caco-2 cells were transduced with lentivirus encoding CRISPR library followed by selection with puromycin. The populations of cells with low TMPRSS2 expression were enriched by FACS with the TMPRSS2-specific antibody, subjected to genomic DNA isolation followed by Illumina sequencing of the integrated gRNAs. (B, C) MAGecK RRA (robust rank aggregation) scores of the enriched sgRNAs from the CRISPR-Cas9 screens performed with the genome-wide sgRNA library (B) and sgRNA library targeting Epigenetic Modifiers and Transcriptional Regulators (C).

Article Snippet: Genetic screens with genome-wide and Epigenetic modifiers and transcriptional regulators CRISPR sgRNA libraries Human Improved Genome-wide Knockout CRISPR Library v1 was a gift from Kosuke Yusa (Addgene #67989)73.

Techniques: Genome Wide, CRISPR, Expressing, Transduction, Selection, DNA Extraction, Illumina Sequencing

Journal: Scientific reports

Article Title: CRISPR-Cas9 genetic screens reveal regulation of TMPRSS2 by the Elongin BC-VHL complex.

doi: 10.1038/s41598-025-95644-0

Figure Lengend Snippet: Fig. 6. CRISPR Cas9-mediated depletion of Elongin B or PHD inhibitor treatment decrease SARS-CoV-2 infection of Calu-3 cells. (A) Calu-3 Cas9 cells stably expressing sgRNAs targeting b2m, Elongin B (TCEB2) or control sgRNAs were lysed and analysed by immunoblot with antibodies specific for HIF1⍺, HIF2⍺, Elongin B, VHL and b-actin. Asterisks denote non-specific bands. (B) Calu-3 Cas9 cells stably expressing two independent pairs of sgRNAs targeting Elongin B (TCEB2) or control sgRNAs were infected with rSARS- CoV-2 Venus at an MOI of 0.1, harvested 24 h later and subjected to RNA extraction followed by RT-qPCR analysis with the primers specific for SARS-CoV-2 nucleocapsid RNA and 18S. Data are presented as mean of n = 3 technical replicates ± s.d. The statistical significance was assessed by unpaired two-tailed t test. (C) Calu-3 cells were treated with 100 uM FG-4592 (roxadustat) or DMSO as a control for 72 h, infected with rSARS-CoV-2 Venus or Omicron BA.2 at an MOI of 0.1, harvested 24 h later and subjected to RNA extraction followed by RT-qPCR analysis with the primers specific for SARS-CoV-2 nucleocapsid RNA and 18S. Data are presented as mean of n = 3 technical replicates ± s.d. The statistical significance was assessed by unpaired two-tailed t test. (D) Depletion of Elongin B induces downregulation of ACE2 and TMPRSS2 in Calu-3 Cas9 cells. The cells from the panel 6A (right) were subjected to RNA isolation followed by RT-qPCR analysis with primers specific for TMPRSS2, ACE2, PGK1, CA9 and 18S. Data are presented as mean of n = 3 technical replicates ± s.d. The statistical significance was assessed by unpaired two-tailed t test. (E) PHD inhibitor treatment induces downregulation of ACE2 and TMPRSS2 in Calu-3 cells. The cells were treated with 100 uM FG-4592 (roxadustat) for 72 h and subjected to RT-qPCR analysis as described above (6D).

Article Snippet: Genetic screens with genome-wide and Epigenetic modifiers and transcriptional regulators CRISPR sgRNA libraries Human Improved Genome-wide Knockout CRISPR Library v1 was a gift from Kosuke Yusa (Addgene #67989)73.

Techniques: CRISPR, Infection, Stable Transfection, Expressing, Control, Western Blot, RNA Extraction, Quantitative RT-PCR, Two Tailed Test, Isolation

Journal: eLife

Article Title: SOD1 is a synthetic-lethal target in PPM1D -mutant leukemia cells

doi: 10.7554/eLife.91611

Figure Lengend Snippet: ( A ) Immunoblot validation of PPM1D -mutant Cas9-expressing OCI-AML2 cells generated and used for CRISPR screening. Blots were probed with anti-PPM1D (1:1000) and GAPDH (1:1000). Clones 2102 and 2113 were selected for the dropout screen. ( B ) Venn diagram of genes that were depleted from the two PPM1D -mutant clones (#2102, 2113) used in the dropout screen, but not depleted in the wild-type (WT) control lines. 37 genes were found to be depleted in both mutant clones. For a full list of genes, see . ( C ) Volcano plot of synthetic-lethal hits ranked by fitness score with the Fanconi anemia pathway genes highlighted in blue. ( D ) Immunoblot validation of SOD1 deletion. WT and PPM1D -mutant Cas9-OCI-AML2 cells were transduced with control (empty vector [EV]) or sg SOD1 lentiviruses. Two sgRNAs targeting SOD1 were tested. Three days post-transduction, the cells underwent puromycin selection (3µg/mL) for 3 days after which they were harvested for western blot. Blots were probed with anti-PPM1D (1:1000), anti-SOD1 (1:500), and anti-vinculin (1:2500). ( E ) Cas9-OCI-AML2 and Cas9-OCI-AML3 WT or PPM1D -mutant cells were transduced with the empty vector control backbone tagged with a blue fluorescent protein (BFP) reporter. Cells were assayed by flow cytometry between 3 and 24 days post-transduction and normalized to the BFP percentage at day 3. Data shown are mean ± SD (n=2 per condition). Figure 1—figure supplement 1—source data 1. Western blot validation of OCI-AML2 PPM1D-mutant clones after CRISPR editing. Figure 1—figure supplement 1—source data 2. Western blot validation of SOD1 deletion in WT and PPM1D-mutant cells.

Article Snippet: For large-scale production of lentivirus, 15 cm plates of 80–90% confluent 293T cells were transfected using Lipofectamine 2000 (Invitrogen) with 7.5 μg of the Human Improved Whole-Genome Knockout CRISPR library V1 (by Kosuke Yuya, Addgene #67989), 18.5 μg of psPax2, and 4 μg of pMD2.G.

Techniques: Western Blot, Biomarker Discovery, Mutagenesis, Expressing, Generated, CRISPR, Clone Assay, Control, Transduction, Plasmid Preparation, Selection, Flow Cytometry

Journal: eLife

Article Title: SOD1 is a synthetic-lethal target in PPM1D -mutant leukemia cells

doi: 10.7554/eLife.91611

Figure Lengend Snippet: ( A ) Schematic of whole-genome CRISPR dropout screen. Wild-type (WT) Cas9-expressing OCI-AML2 and two isogenic PPM1D -mutant lines were transduced with the Human Improved Whole Genome Knockout CRISPR library V1 containing 90,709 guide RNAs (gRNAs) targeting 18,010 human genes at low multiplicity of infection (MOI~0.3). Each condition was performed in technical triplicates. Three days post-transduction, cells underwent puromycin selection for 3 days. Cells were harvested at day 10 as the initial timepoint and then harvested every 3 days afterward. sgRNA-sequencing was performed on cells collected on day 28. ( B ) Top biological processes based on gene ontology analysis of the top 37 genes essential for PPM1D -mutant cell survival. Enrichment and depletion of guides and genes were analyzed using MAGeCK-VISPR by comparing read counts from each PPM1D -mutant cell line replicate with counts from the initial starting population at day 10. ( C ) Volcano plot of synthetic-lethal hits ranked by fitness score with a negative score indicating genes for which their knockout leads to decreased growth or survival. SOD1 (highlighted) was the top hit from the screen. ( D ) Left: Schematic of competitive proliferation assays used for validation of CRISPR targets. Right: WT and PPM1D- mutant Cas9-OCI-AML2 and Cas9-OCI-AML3 cells were transduced with lentiviruses containing a single SOD1 -gRNA with a blue fluorescent protein (BFP) reporter. Cells were assayed by flow cytometry every 3–4 days and normalized to the BFP percentage at day 3 post-transduction. Two unique gRNAs against SOD1 were used per cell line and each condition was performed in technical duplicates; multiple unpaired t-tests, **p<0.01, ***p<0.001. ( E ) Left: Cas9-expressing WT and PPM1D -mutant cells were transduced with control or sg SOD1 -containing lentiviruses and underwent puromycin (3 µg/mL) selection for 3 days prior to transplantation. Sublethally irradiated (250 cGy) NSG mice were intravenously transplanted with 3×10 6 cells. Right: Kaplan-Meier survival curve of mice transplanted with WT or PPM1D -mutant (gray) leukemia cells with or without SOD1 deletion. The median survival of mice transplanted with WT, WT/ SOD1 –/– , PPM1D mut , and PPM1D mut / SOD1 –/– leukemia cells was 32, 43, 32, and 55 days, respectively; Mantel-Cox test, **p<0.01, ***p<0.001. Figure 1—source data 1. CRISPR dropout screen raw data and top 37 gene candidates.

Article Snippet: For large-scale production of lentivirus, 15 cm plates of 80–90% confluent 293T cells were transfected using Lipofectamine 2000 (Invitrogen) with 7.5 μg of the Human Improved Whole-Genome Knockout CRISPR library V1 (by Kosuke Yuya, Addgene #67989), 18.5 μg of psPax2, and 4 μg of pMD2.G.

Techniques: CRISPR, Expressing, Mutagenesis, Transduction, Knock-Out, Infection, Selection, Sequencing, Biomarker Discovery, Flow Cytometry, Control, Transplantation Assay, Irradiation

Journal: eLife

Article Title: SOD1 is a synthetic-lethal target in PPM1D -mutant leukemia cells

doi: 10.7554/eLife.91611

Figure Lengend Snippet: ( A ) Left: Sanger sequencing traces of the parental U2OS cell line harboring a c.1372 C>T mutation in PPM1D and the CRISPR-edited U2OS cell line with mutation corrected to wild-type (WT) PPM1D. Right: Immunoblot validation of these clones are shown. Lysates were probed with anti-PPM1D (1:1000) and anti-GAPDH (1:1000). ( B,C ) Left: Representative images of Rad51 and 53BP1 immunofluorescence microscopy. Mouse embryonic fibroblasts were treated with 10 Gy irradiation, harvested 1 hr post-irradiation and stained for the indicated markers. Right: Quantification of the number of foci per cell is shown. Analysis was performed using CellProfiler. n>100 cells for each condition; Student’s t-test. ( D ) Comet assay quantification of mouse embryonic fibroblasts at baseline and after 1 hr post-irradiation (10 Gy). Quantification and analyses of tail moments were performed using the Comet IV software. n≥150 comets were scored per experimental group; two-way ANOVA, ns = non-significant (p>0.05), *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. Figure 5—figure supplement 1—source data 1. Western blot analysis of CRISPR-edited U2OS clones validating the correction of the endogenous PPM1D mutations to the wild type form. Figure 5—figure supplement 1—source data 2. Immunofluorescence microscopy of WT and Ppm1d-mutant mouse embryonic fibroblasts stained with Rad51. Figure 5—figure supplement 1—source data 3. Immunofluorescence microscopy of WT and Ppm1d-mutant mouse embryonic fibroblasts stained with 53BP1.

Article Snippet: For large-scale production of lentivirus, 15 cm plates of 80–90% confluent 293T cells were transfected using Lipofectamine 2000 (Invitrogen) with 7.5 μg of the Human Improved Whole-Genome Knockout CRISPR library V1 (by Kosuke Yuya, Addgene #67989), 18.5 μg of psPax2, and 4 μg of pMD2.G.

Techniques: Sequencing, Mutagenesis, CRISPR, Western Blot, Biomarker Discovery, Clone Assay, Immunofluorescence, Microscopy, Irradiation, Staining, Single Cell Gel Electrophoresis, Software