genome wide crispr knockout pooled library screen human geckov2 crispr knockout pooled libraries  (Addgene inc)

Journal: Genome biology

Article Title: Systematic interrogation of functional genes underlying cholesterol and lipid homeostasis.

doi: 10.1186/s13059-025-03531-8

Figure Lengend Snippet: Fig. 1 Synthetic lethal CRISPR screening to identify potential cholesterol regulators in HepG2 cells. A Immunoblot analysis of HMGCR in HepG2 cells undergoing CRISPR-mediated gene knockout with two independent sgRNAs (numbered as _1 and _2). Vector without specific sgRNA insert serves as a control. GAPDH serves as a loading control. B Immunoblot analysis of LDLR in HepG2 cells that have undergone CRISPR-mediated gene knockout with two independent sgRNAs. C The cell growth analysis of HepG2 cells after introducing indicated sgRNAs via lentiviral infection for 7 days. Cells were counted with a hemacytometer. Mean ± SD with n = 3. Ordinary one-way ANOVA with Tukey’s test, **p < 0.01, ***p < 0.001. D The relative cell viability was determined by CCK-8 assay for HepG2 cells expressing indicated sgRNAs and treated with indicated doses of lovastatin. Mean ± SD with n = 6. Ordinary one-way ANOVA with Dunnett’s test, *p < 0.05, **p < 0.01, ***p < 0.001. E The workflow of genome-scale synthetic lethal CRISPR screens (Screen 1) to identify negative GIs with HMGCR using its inhibitor lovastatin in HepG2 cells. F The scatter plot showing the β score of each gene and the correlation of both CRISPR screens (vehicle and lovastatin) in HepG2 cells. The genes in blue box are preferential targets as the synthetic lethal or negative GI hits. G The rank-ordered list of each gene in the CRISPR screens according to the strength of synthetic lethality measured by differential β scores between lovastatin and vehicle conditions. The top interesting gene hits are highlighted. H The top selected functional terms enriched among synthetic lethal hits of the CRISPR screens (Screen 1) as determined by the gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis

Article Snippet: The genome-wide CRISPR knockout screens were performed with a human H3 CRISPR knockout library (Addgene pooled library, 133914) which was designed with improved algorithms and superior performance.

Techniques: CRISPR, Western Blot, Gene Knockout, Plasmid Preparation, Control, Infection, CCK-8 Assay, Expressing, Functional Assay

Journal: Genome biology

Article Title: Systematic interrogation of functional genes underlying cholesterol and lipid homeostasis.

doi: 10.1186/s13059-025-03531-8

Figure Lengend Snippet: Fig. 2 Multiple CRISPR screens in HeLa cells for cholesterol regulatory genes. A The cell growth analysis of HeLa cells after introducing indicated sgRNAs via lentiviral infection for 7 days. Cells were counted with a hemacytometer. Mean ± SD with n = 3. Ordinary one-way ANOVA with Tukey’s test, ***p < 0.001. B The relative cell growth determined by cell counting for HeLa cells introducing indicated sgRNAs and treated with indicated doses of lovastatin. Mean ± SD with n = 3. Ordinary one-way ANOVA with Dunnett’s test, **p < 0.01, ***p < 0.001. C The workflow of genome-scale synthetic lethal CRISPR screens (Screen 2) to identify negative GIs with HMGCR using its inhibitor lovastatin in HeLa cells. D The scatter plot showing the β score of each gene and the correlation of both CRISPR screens (vehicle and lovastatin) in HeLa cells. The genes in blue box are preferential targets as the synthetic lethal or negative GI hits. E The top selected functional terms enriched among synthetic lethal hits of the CRISPR screens (Screen 2) as determined by the GO and KEGG analysis. F The workflow of genome-scale synthetic lethal CRISPR screens (Screen 3) to identify negative GIs with LDLR in LDLR KO single clone and AAVS1 KO control HeLa cells. G The rank-ordered list of each gene in the CRISPR screens (Screen 3) according to the strength of synthetic lethality measured by differential tRRA scores between LDLR KO single clone and AAVS1 KO control HeLa cells. The top interesting gene hits are highlighted. H Venn diagram showing the overlap of synthetic lethal or negative GI hits between the two independent LDLR KO clones in the CRISPR screens (Screen 3). I Venn diagram showing the overlap of synthetic lethal or negative GI hits between the three CRISPR screens. J Heatmap showing the relative strength of GIs of top selected hits to HMGCR or LDLR across different CRISPR screens

Article Snippet: The genome-wide CRISPR knockout screens were performed with a human H3 CRISPR knockout library (Addgene pooled library, 133914) which was designed with improved algorithms and superior performance.

Techniques: CRISPR, Infection, Cell Counting, Functional Assay, Control, Clone Assay

Journal: Genome biology

Article Title: Systematic interrogation of functional genes underlying cholesterol and lipid homeostasis.

doi: 10.1186/s13059-025-03531-8

Figure Lengend Snippet: Fig. 4 Integrative multi-omics analysis to pinpoint key cholesterol regulators. A Heatmap showing the differentially expressed proteins in LDLR/HMGCR DKO cells compared to Vector control HepG2 cells determined by mass spectrometry-based proteomics profiling. The number of up- or down-regulated proteins is indicated along the heatmap. B Venn diagram showing the overlap of either up-regulated or down-regulated genes in LDLR/HMGCR DKO cells compared to Vector control HepG2 cells between RNA-seq and proteomics analysis. C Volcano plot showing the differentially expressed proteins in LDLR/HMGCR DKO cells compared to control HepG2 cells with several typical proteins highlighted. D The top ten enriched functional terms for up-regulated proteins in LDLR/HMGCR DKO cells compared to control HepG2 cells by GO and KEGG analysis. E The analytic scheme of human genes associated with lipid disorders or cardiovascular diseases. HDL: high-density lipoprotein; LDL: low-density lipoprotein; VLDL: very low-density lipoprotein; TC: total cholesterol; TG: triglycerides; CAD: coronary artery disease. F Venn diagram showing the overlap of potential cholesterol or lipid regulators between different angles including synthetic lethal CRISPR screens (Screen 1–3), DEGs in RNA-seq analysis (DKO vs. vector in HepG2 and HeLa cells, sterol depleted vs normal, and SREBF2 KO vs vector), differentially expressed proteins in proteomics analysis (DKO vs. vector in HepG2 cells), and the compiled list of human genes with variants related to lipid disorders or related diseases. The genes of indicated intersections are highlighted

Article Snippet: The genome-wide CRISPR knockout screens were performed with a human H3 CRISPR knockout library (Addgene pooled library, 133914) which was designed with improved algorithms and superior performance.

Techniques: Biomarker Discovery, Plasmid Preparation, Control, Mass Spectrometry, RNA Sequencing, Functional Assay, CRISPR

Journal: Genome biology

Article Title: Systematic interrogation of functional genes underlying cholesterol and lipid homeostasis.

doi: 10.1186/s13059-025-03531-8

Figure Lengend Snippet: Fig. 5 Functions of GGT7 during cholesterol and lipid homeostasis. A RNA expression analysis of indicated genes by RT-qPCR in HepG2 cells upon LDLR KO, HMGCR KO, or LDLR/HMGCR DKO. Mean ± SD with n = 3. Ordinary one-way ANOVA with Dunnett’s test, compared to Vector control, **p < 0.01, ***p < 0.001. B RNA expression analysis of indicated genes by RT-qPCR in HepG2 cells upon SREBF2 KO under sterol deprivation condition. Mean ± SD with n = 3. Unpaired two-sided t test, compared to vector control, **p < 0.01, ***p < 0.001. C Immunoblot analysis of GGT7 in HepG2 cells undergoing CRISPR-mediated gene knockout with two independent sgRNAs. GAPDH serves as a loading control. D Decreased total cholesterol levels of HepG2 cells upon GGT7 KO using two independent sgRNAs. Mean ± SD with n = 3. Unpaired two-sided t test, compared to vector control, *p < 0.05. E Decreased sterol and other lipids upon GGT7 KO in HepG2 cells determined by untargeted metabolomic profiling. n = 6 biological replicates for each group

Article Snippet: The genome-wide CRISPR knockout screens were performed with a human H3 CRISPR knockout library (Addgene pooled library, 133914) which was designed with improved algorithms and superior performance.

Techniques: RNA Expression, Quantitative RT-PCR, Plasmid Preparation, Control, Western Blot, CRISPR, Gene Knockout

Journal: Genome biology

Article Title: Systematic interrogation of functional genes underlying cholesterol and lipid homeostasis.

doi: 10.1186/s13059-025-03531-8

Figure Lengend Snippet: Fig. 6 Mechanistic insights of GGT7 in regulating cholesterol metabolism. A RNA expression analysis of indicated genes by RT-qPCR in HepG2 cells upon GGT7 KO. Mean ± SD with n = 3. Unpaired two-sided t test, compared to Vector control, *p < 0.05, **p < 0.01, ***p < 0.001. B RNA expression analysis of indicated genes by RT-qPCR in HepG2 cells upon GGT7 KO under normal or sterol deprivation conditions. Mean ± SD with n = 3. Unpaired two-sided t test, *p < 0.05, **p < 0.01, ***p < 0.001. C Volcano plot showing the DEGs in GGT7 KO cells compared to vector control HepG2 cells with several typical genes highlighted. The number of up- or down-regulated DEGs is indicated. D The top selected functional terms enriched for down-regulated DEGs in GGT7 KO cells compared to control HepG2 cells by GO and KEGG analysis. E Coomassie blue staining of SDS-PAGE gel with FLAG bead immunoprecipitated materials from vector control- or FLAG-GGT7-expressing HepG2 cells. The band position corresponding to GGT7 or MYH10 is indicated with an asterisk. F The top ten list of GGT7-interacting protein partners identified by mass spectrometry. G Immunoblot analysis of total cell lysis and immunoprecipitants (using IgG control, GGT7, or MYH10 antibody) for indicated proteins derived from HepG2 cells. H Immunoblot analysis of MYH10 in HepG2 cells that have undergone CRISPR-mediated gene knockout. GAPDH serves as a loading control. I Decreased total cholesterol levels of HepG2 cells upon MYH10 knockout. Mean ± SD with n = 3. Unpaired two-sided t test, compared to AAVS1 KO, *p < 0.05. Decreased Dil-LDL uptake by HepG2 cells upon J GGT7 or K MYH10 knockout. Mean ± SD with n = 3. Unpaired two-sided t test, compared to AAVS1 KO, **p < 0.01, ***p < 0.001

Article Snippet: The genome-wide CRISPR knockout screens were performed with a human H3 CRISPR knockout library (Addgene pooled library, 133914) which was designed with improved algorithms and superior performance.

Techniques: RNA Expression, Quantitative RT-PCR, Plasmid Preparation, Control, Functional Assay, Staining, SDS Page, Immunoprecipitation, Expressing, Mass Spectrometry, Western Blot, Lysis, Derivative Assay, CRISPR, Gene Knockout, Knock-Out