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: Cell Reports

Article Title: Rare Variant Burden Analysis within Enhancers Identifies CAV1 as an ALS Risk Gene

doi: 10.1016/j.celrep.2020.108456

Figure Lengend Snippet:

Article Snippet: Alt-R CRISPR-Cas9 Control Kit, Human, 2 nmol , Integrated DNA technologies , #1072554.

Techniques: Recombinant, Electroporation, Modification, Reverse Transcription, SYBR Green Assay, Random Hexamer, Protease Inhibitor, Western Blot, Saline, Plasmid Preparation, BIA-KA, Transfection, CRISPR, Control, Software, Imaging