human crispr metabolic gene knockout library  (Addgene inc)

Journal: Nature

Article Title: Targeting PIKfyve-driven lipid metabolism in pancreatic cancer.

doi: 10.1038/s41586-025-08917-z

Figure Lengend Snippet: Fig. 3 | PIKfyve inhibition obligates PDAC cells to stimulate a lipogenic transcriptional and metabolic program. a, Schematic of the metabolism- focused CRISPR screen in MIA PaCa-2 cells. Created in BioRender. Cheng, C. (2025) https://BioRender.com/d149928. b,c, Gene enrichment rank plot-based differential sgRNA representation (b) and scatter plot of gene fitness scores (c) of high dose (2,000 nM) and low dose (100 nM) apilimod-treated versus DMSO- treated end-point populations of the CRISPR screen experiment. Graphs show the top 30 synthetically lethal genes involved in fatty acid and sphingolipid synthesis (red), the top 30 synthetically lethal genes involved in cholesterol synthesis (purple) and genes that confer sensitivity to apilimod (blue). d, Metabolic map of sphingolipid and cholesterol synthesis. The figure shows the top 90 synthetically lethal genes involved in fatty acid and sphingolipid

Article Snippet: The human CRISPR metabolic gene knockout library was a gift from David Sabatini (Addgene, 110066)58.

Techniques: Inhibition, CRISPR

Journal: Science Advances

Article Title: Lipoylation inhibition enhances radiation control of lung cancer by suppressing homologous recombination DNA damage repair

doi: 10.1126/sciadv.adt1241

Figure Lengend Snippet: ( A ) Schematic illustration of CRISPR-Cas9 screen workflow. ( B ) Top 10 negatively selected genes ranked by robust rank aggregation (RRA) score in 10 Gy–irradiated group compared to the control. ( C ) Manhattan plot of the entire 2981 metabolic genes by log 10 P value. The top 10 negatively selected genes are highlighted. ( D ) Schematic illustration of the function of LIPT1, LIAS, DLD, and PDHX, the four top hits involved in lipoylation. ( E ) Pathway analysis of the 10 most significantly depleted metabolic pathways by Gene Ontology classification in 10 Gy–irradiated cells compared to the nonirradiated cells. ( F ) Immunoblots of total and lipoylated PDH and α-KGDH subunits (DLAT and DLST, respectively). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), DLAT, and DLST were blotted as loading controls. ( G ) Clonogenic assay of indicated cell lines after 2, 4, 6, and 8 Gy IR. The surviving fraction was normalized to the corresponding sham control, and survival curves were fitted using the linear-quadratic model.

Article Snippet: The human CRISPR knockout pooled library was designed to target 2981 metabolic genes (Addgene, #110066) ( ) and synthesized as a pool of lentiviral vectors containing unique gRNA sequences ( ).

Techniques: CRISPR, Irradiation, Control, Western Blot, Clonogenic Assay

Journal: Cell reports

Article Title: SLC7A5 is required for cancer cell growth under arginine-limited conditions.

doi: 10.1016/j.celrep.2024.115130

Figure Lengend Snippet: Figure 1. A functional genomics screen identifies SLC7A5 as required for growth on citrulline (A) Schematic of citrulline synthesis into arginine. (B) Concentrations of arginine and citrulline in mouse serum (mean ± SD, n = 5). (C) Rates of appearance of arginine and citrulline as determined by steady-state labeling fraction upon defined infusion (mean ± SD, n = 5). (D) Media conditions used for the CRISPR screen. (E) Growth of A375mASS1-OE cells in Low Arg, High Arg, and Low Arg minus Cit media. Data are expressed in terms of relative growth, where the readings on day 0 are normalized to 1 (mean ± SD, n = 4). (F) Schematic of the CRISPR-based screen to identify metabolic genes required for growth under low-arginine conditions. (G) Gene scores from cells grown under Low Arg vs. High Arg. SLC7A5, the top hit, is highlighted in red. SLC7A1 (arginine transporter) and ASL (arginosuccinate lyase) are also highlighted. The red line is the equation y = x passing though (0,0) to highlight differential essentiality. (H) Top 25 genes scoring as selectively essential in Low Arg vs. High Arg. Genes linked to glycosylation are shown in blue, the urea cycle in red, transport in purple, reactive oxygen species (ROS) metabolism in orange, and other genes in green.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Oligonucleotides sgRNA for CRISPR KO and qPCR Oligonucleotides This study Table S1 Recombinant DNA Human ASS1 in pLX304 DNASU Cat#HSCD00438196 Human SLC7A5 in pDONR221 DNASU Cat#HSCD00042452 pLX307 Empty Vector Addgene Cat#41392 Human SLC7A5 in pLX307 This study N/A pSpCas9(BB)-2A-GFP (PX458)-ASS1_exon11 This study N/A pSpCas9(BB)-2A-Puro (PX459) V2.0-SLC7A5_exon2 This study N/A pSpCas9(BB)-2A-Puro (PX459) V2.0 Addgene Cat#62988 pSpCas9(BB)-2A-GFP (PX458) Addgene Cat#48138 Human CRISPR Metabolic Gene Knockout Library Addgene Cat#110066 psPAX2 Addgene Cat#12260 PMD2.G Addgene Cat#12259 Software and algorithms MAGeCK GitHub https://github.com/liulab-dfci/MAGeCK GraphPad Prism 10 GraphPad Software https://www.graphpad.com/ R Version 4.4.0 R https://cran.r-project.org/ EL-MAVEN Software Elucidata Agrawal et al.79 AccuCor GitHub https://github.com/XiaoyangSu/AccuCor Biorender Biorender https://www.biorender.com/ Other Countess 3 Automated Cell Counter Invitrogen Cat#AMQAX2000 BioTek Synergy Neo2 Hybrid Multimode Reader Agilent N/A BioRad ChemiDoc MP Imaging System BioRad Cat#12003154 Cryomill Retsch N/A LC480 PCR Lightcycler Roche Cat#05015278001

Techniques: Functional Assay, Labeling, CRISPR, Glycoproteomics

Journal: Molecular Metabolism

Article Title: Acetate drives ovarian cancer quiescence via ACSS2-mediated acetyl-CoA production

doi: 10.1016/j.molmet.2024.102031

Figure Lengend Snippet: Multi-omics analysis identifies ACSS2 as a candidate regulator of quiescence . (A – B) Schematic representation of RNA-Seq (A) and CRISPR KO screen (B) of serum starved (serum free) Ovcar8 cells. (C) Cross-comparison of RNA-seq and CRISPR KO screen results (cutoffs for RNA-seq: fold-change ≥2, FDR ≤0.1; Cutoff for CRISPR screen: pval ≤0.1). (D) Volcano plot of RNA-seq of serum starved Ovcar8 results showing differential expression of common genes from (C) . (E) ACSS2 protein expression was determined by western blotting in the indicated cells. β-actin was used as a loading control. Western blots shown are representative data from at least 2 independent experiments in each cell line and condition.

Article Snippet: Human pooled metabolic CRISPR KO library (Addgene #110066), pLV-mCherry-hCdt1(1–100)ΔCy (Addgene #193759), and pCDH-EF1-mVenus-p27K − (Addgene #176651) were obtained from Addgene.

Techniques: Biomarker Discovery, RNA Sequencing, CRISPR, Comparison, Quantitative Proteomics, Expressing, Western Blot, Control