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human crispr metabolic gene knockout library addgene  (Addgene inc)


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    Addgene inc human crispr metabolic gene knockout library addgene
    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 <t>CRISPR</t> 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 <t>identify</t> <t>metabolic</t> 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.
    Human Crispr Metabolic Gene Knockout Library Addgene, supplied by Addgene inc, used in various techniques. Bioz Stars score: 96/100, based on 4079 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/human+crispr+metabolic+gene+knockout+library+addgene/pSpCas9(BB)-2A-GFP+(PX458)+(Plasmid+%2348138)/pm39756034-230-63-69
    Average 96 stars, based on 4079 article reviews
    human crispr metabolic gene knockout library addgene - by Bioz Stars, 2026-09
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    1) Product Images from "SLC7A5 is required for cancer cell growth under arginine-limited conditions."

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

    Journal: Cell reports

    doi: 10.1016/j.celrep.2024.115130

    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.
    Figure Legend 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.

    Techniques Used: Functional Assay, Labeling, CRISPR, Glycoproteomics

    Related Articles

    CRISPR:

    Article Title: SLC7A5 is required for cancer cell growth under arginine-limited conditions.
    Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER 2-Mercaptoethanol Sigma-Aldrich Cat#M6250 Proteinase K New England Biolabs Cat#P8107S Trypan Blue 0.4% ThermoFisher Cat#15250061 PMSF Cell Signaling Cat#8553S Polybrene Sigma Aldrich Cat#TR-1003 Poly-D-Lysine ThermoFisher Cat#A3890401 Lipofectamine 3000 Transfection Reagent ThermoFisher Cat#L3000008 L-Amino Acid Diet Research Diets Cat#A10021Bi L-Amino Acid Diet -Arginine Research Diets Cat#A10036i qScript cDNA SuperMix VWR Cat#95048 Resazurin sodium salt Sigma-Aldrich Cat#R7017 SYBR Green Master Mix ThermoFisher Cat#A46109 NEBNext Ultra II Q5 Master Mix New England Biolabs Cat#M0544L [1-13C]Citrulline Cambridge Isotopes Cat#CLM-4899 [U-13C]Arginine Cambridge Isotopes Cat#CLM-2265 XTremeGene 9 Sigma-Aldrich Cat#6365779001 Phosphate Buffered Saline, pH 7.4 ThermoFisher Cat#10010031 Critical commercial assays Pierce BCA Protein Assay Kit Thermo Scientific Cat#23227 Pierce Rapid Gold BCA Protein Assay Kit Thermo Scientific Cat#A52335 Trans Blot Turbo RTA Transfer Kit BioRad Cat#1704270 QIAprep Spin Miniprep Kit Qiagen Cat#27106 RNAeasy Mini Kit Qiagen Cat#74104 DNeasy Blood & Tissue Kit Qiagen Cat#69504 QIAmp DNA Blood Maxi Kit Qiagen Cat#51192 Mycoplasma PCR Detection Kit ABM Cat#G238 BioRad Gels 4–20% BioRad Cat#4561096 Deposited data Metabolite Abundance NMDR ST003613 Experimental models: Cell lines Human: A375m Roh-Johnson Lab, University of Utah N/A Human: HCT116 Rabinowitz Lab, Princeton N/A Human: HCT15 Rutter Lab, U of U N/A Human: HEK-293FT Roh-Johnson Lab, U of U N/A Human: LN229 Rabinowitz Lab, Princeton N/A Human: MCF7 Rutter Lab, U of U N/A Human: MDA-MB-468 Roh-Johnson Lab, U of U N/A Human: NCI-H460 McMahon Lab, U of U N/A Human: RPMI7951 ATCC Cat#HTB-66 Human: MeWo Rutter Lab, U of U N/A Human: MM253 Rutter Lab, U of U N/A Human: SKMEL28 Rabinowitz Lab, Princeton N/A Mouse: EO771 Hilgendorf Lab, U of U N/A Experimental models: Organisms/strains NOD.Cg-Rag1tm1Mom Il2rgtm1Wjl/SzJ (NRG) University of Utah Preclinical Research Resource Core N/A Dct-TVA;BrafV600E;Cdkn2alox/lox Grossmann Lab, U of U N/A C57BL6 Charles River Cat#027 (Continued on next page) 16 Cell Reports 44, 115130, January 28, 2025 .. 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 Cat110066 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 ..

    Real-time Polymerase Chain Reaction:

    Article Title: SLC7A5 is required for cancer cell growth under arginine-limited conditions.
    Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER 2-Mercaptoethanol Sigma-Aldrich Cat#M6250 Proteinase K New England Biolabs Cat#P8107S Trypan Blue 0.4% ThermoFisher Cat#15250061 PMSF Cell Signaling Cat#8553S Polybrene Sigma Aldrich Cat#TR-1003 Poly-D-Lysine ThermoFisher Cat#A3890401 Lipofectamine 3000 Transfection Reagent ThermoFisher Cat#L3000008 L-Amino Acid Diet Research Diets Cat#A10021Bi L-Amino Acid Diet -Arginine Research Diets Cat#A10036i qScript cDNA SuperMix VWR Cat#95048 Resazurin sodium salt Sigma-Aldrich Cat#R7017 SYBR Green Master Mix ThermoFisher Cat#A46109 NEBNext Ultra II Q5 Master Mix New England Biolabs Cat#M0544L [1-13C]Citrulline Cambridge Isotopes Cat#CLM-4899 [U-13C]Arginine Cambridge Isotopes Cat#CLM-2265 XTremeGene 9 Sigma-Aldrich Cat#6365779001 Phosphate Buffered Saline, pH 7.4 ThermoFisher Cat#10010031 Critical commercial assays Pierce BCA Protein Assay Kit Thermo Scientific Cat#23227 Pierce Rapid Gold BCA Protein Assay Kit Thermo Scientific Cat#A52335 Trans Blot Turbo RTA Transfer Kit BioRad Cat#1704270 QIAprep Spin Miniprep Kit Qiagen Cat#27106 RNAeasy Mini Kit Qiagen Cat#74104 DNeasy Blood & Tissue Kit Qiagen Cat#69504 QIAmp DNA Blood Maxi Kit Qiagen Cat#51192 Mycoplasma PCR Detection Kit ABM Cat#G238 BioRad Gels 4–20% BioRad Cat#4561096 Deposited data Metabolite Abundance NMDR ST003613 Experimental models: Cell lines Human: A375m Roh-Johnson Lab, University of Utah N/A Human: HCT116 Rabinowitz Lab, Princeton N/A Human: HCT15 Rutter Lab, U of U N/A Human: HEK-293FT Roh-Johnson Lab, U of U N/A Human: LN229 Rabinowitz Lab, Princeton N/A Human: MCF7 Rutter Lab, U of U N/A Human: MDA-MB-468 Roh-Johnson Lab, U of U N/A Human: NCI-H460 McMahon Lab, U of U N/A Human: RPMI7951 ATCC Cat#HTB-66 Human: MeWo Rutter Lab, U of U N/A Human: MM253 Rutter Lab, U of U N/A Human: SKMEL28 Rabinowitz Lab, Princeton N/A Mouse: EO771 Hilgendorf Lab, U of U N/A Experimental models: Organisms/strains NOD.Cg-Rag1tm1Mom Il2rgtm1Wjl/SzJ (NRG) University of Utah Preclinical Research Resource Core N/A Dct-TVA;BrafV600E;Cdkn2alox/lox Grossmann Lab, U of U N/A C57BL6 Charles River Cat#027 (Continued on next page) 16 Cell Reports 44, 115130, January 28, 2025 .. 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 Cat110066 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 ..

    Recombinant:

    Article Title: SLC7A5 is required for cancer cell growth under arginine-limited conditions.
    Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER 2-Mercaptoethanol Sigma-Aldrich Cat#M6250 Proteinase K New England Biolabs Cat#P8107S Trypan Blue 0.4% ThermoFisher Cat#15250061 PMSF Cell Signaling Cat#8553S Polybrene Sigma Aldrich Cat#TR-1003 Poly-D-Lysine ThermoFisher Cat#A3890401 Lipofectamine 3000 Transfection Reagent ThermoFisher Cat#L3000008 L-Amino Acid Diet Research Diets Cat#A10021Bi L-Amino Acid Diet -Arginine Research Diets Cat#A10036i qScript cDNA SuperMix VWR Cat#95048 Resazurin sodium salt Sigma-Aldrich Cat#R7017 SYBR Green Master Mix ThermoFisher Cat#A46109 NEBNext Ultra II Q5 Master Mix New England Biolabs Cat#M0544L [1-13C]Citrulline Cambridge Isotopes Cat#CLM-4899 [U-13C]Arginine Cambridge Isotopes Cat#CLM-2265 XTremeGene 9 Sigma-Aldrich Cat#6365779001 Phosphate Buffered Saline, pH 7.4 ThermoFisher Cat#10010031 Critical commercial assays Pierce BCA Protein Assay Kit Thermo Scientific Cat#23227 Pierce Rapid Gold BCA Protein Assay Kit Thermo Scientific Cat#A52335 Trans Blot Turbo RTA Transfer Kit BioRad Cat#1704270 QIAprep Spin Miniprep Kit Qiagen Cat#27106 RNAeasy Mini Kit Qiagen Cat#74104 DNeasy Blood & Tissue Kit Qiagen Cat#69504 QIAmp DNA Blood Maxi Kit Qiagen Cat#51192 Mycoplasma PCR Detection Kit ABM Cat#G238 BioRad Gels 4–20% BioRad Cat#4561096 Deposited data Metabolite Abundance NMDR ST003613 Experimental models: Cell lines Human: A375m Roh-Johnson Lab, University of Utah N/A Human: HCT116 Rabinowitz Lab, Princeton N/A Human: HCT15 Rutter Lab, U of U N/A Human: HEK-293FT Roh-Johnson Lab, U of U N/A Human: LN229 Rabinowitz Lab, Princeton N/A Human: MCF7 Rutter Lab, U of U N/A Human: MDA-MB-468 Roh-Johnson Lab, U of U N/A Human: NCI-H460 McMahon Lab, U of U N/A Human: RPMI7951 ATCC Cat#HTB-66 Human: MeWo Rutter Lab, U of U N/A Human: MM253 Rutter Lab, U of U N/A Human: SKMEL28 Rabinowitz Lab, Princeton N/A Mouse: EO771 Hilgendorf Lab, U of U N/A Experimental models: Organisms/strains NOD.Cg-Rag1tm1Mom Il2rgtm1Wjl/SzJ (NRG) University of Utah Preclinical Research Resource Core N/A Dct-TVA;BrafV600E;Cdkn2alox/lox Grossmann Lab, U of U N/A C57BL6 Charles River Cat#027 (Continued on next page) 16 Cell Reports 44, 115130, January 28, 2025 .. 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 Cat110066 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 ..

    Gene Knockout:

    Article Title: SLC7A5 is required for cancer cell growth under arginine-limited conditions.
    Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER 2-Mercaptoethanol Sigma-Aldrich Cat#M6250 Proteinase K New England Biolabs Cat#P8107S Trypan Blue 0.4% ThermoFisher Cat#15250061 PMSF Cell Signaling Cat#8553S Polybrene Sigma Aldrich Cat#TR-1003 Poly-D-Lysine ThermoFisher Cat#A3890401 Lipofectamine 3000 Transfection Reagent ThermoFisher Cat#L3000008 L-Amino Acid Diet Research Diets Cat#A10021Bi L-Amino Acid Diet -Arginine Research Diets Cat#A10036i qScript cDNA SuperMix VWR Cat#95048 Resazurin sodium salt Sigma-Aldrich Cat#R7017 SYBR Green Master Mix ThermoFisher Cat#A46109 NEBNext Ultra II Q5 Master Mix New England Biolabs Cat#M0544L [1-13C]Citrulline Cambridge Isotopes Cat#CLM-4899 [U-13C]Arginine Cambridge Isotopes Cat#CLM-2265 XTremeGene 9 Sigma-Aldrich Cat#6365779001 Phosphate Buffered Saline, pH 7.4 ThermoFisher Cat#10010031 Critical commercial assays Pierce BCA Protein Assay Kit Thermo Scientific Cat#23227 Pierce Rapid Gold BCA Protein Assay Kit Thermo Scientific Cat#A52335 Trans Blot Turbo RTA Transfer Kit BioRad Cat#1704270 QIAprep Spin Miniprep Kit Qiagen Cat#27106 RNAeasy Mini Kit Qiagen Cat#74104 DNeasy Blood & Tissue Kit Qiagen Cat#69504 QIAmp DNA Blood Maxi Kit Qiagen Cat#51192 Mycoplasma PCR Detection Kit ABM Cat#G238 BioRad Gels 4–20% BioRad Cat#4561096 Deposited data Metabolite Abundance NMDR ST003613 Experimental models: Cell lines Human: A375m Roh-Johnson Lab, University of Utah N/A Human: HCT116 Rabinowitz Lab, Princeton N/A Human: HCT15 Rutter Lab, U of U N/A Human: HEK-293FT Roh-Johnson Lab, U of U N/A Human: LN229 Rabinowitz Lab, Princeton N/A Human: MCF7 Rutter Lab, U of U N/A Human: MDA-MB-468 Roh-Johnson Lab, U of U N/A Human: NCI-H460 McMahon Lab, U of U N/A Human: RPMI7951 ATCC Cat#HTB-66 Human: MeWo Rutter Lab, U of U N/A Human: MM253 Rutter Lab, U of U N/A Human: SKMEL28 Rabinowitz Lab, Princeton N/A Mouse: EO771 Hilgendorf Lab, U of U N/A Experimental models: Organisms/strains NOD.Cg-Rag1tm1Mom Il2rgtm1Wjl/SzJ (NRG) University of Utah Preclinical Research Resource Core N/A Dct-TVA;BrafV600E;Cdkn2alox/lox Grossmann Lab, U of U N/A C57BL6 Charles River Cat#027 (Continued on next page) 16 Cell Reports 44, 115130, January 28, 2025 .. 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 Cat110066 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 ..

    Software:

    Article Title: SLC7A5 is required for cancer cell growth under arginine-limited conditions.
    Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER 2-Mercaptoethanol Sigma-Aldrich Cat#M6250 Proteinase K New England Biolabs Cat#P8107S Trypan Blue 0.4% ThermoFisher Cat#15250061 PMSF Cell Signaling Cat#8553S Polybrene Sigma Aldrich Cat#TR-1003 Poly-D-Lysine ThermoFisher Cat#A3890401 Lipofectamine 3000 Transfection Reagent ThermoFisher Cat#L3000008 L-Amino Acid Diet Research Diets Cat#A10021Bi L-Amino Acid Diet -Arginine Research Diets Cat#A10036i qScript cDNA SuperMix VWR Cat#95048 Resazurin sodium salt Sigma-Aldrich Cat#R7017 SYBR Green Master Mix ThermoFisher Cat#A46109 NEBNext Ultra II Q5 Master Mix New England Biolabs Cat#M0544L [1-13C]Citrulline Cambridge Isotopes Cat#CLM-4899 [U-13C]Arginine Cambridge Isotopes Cat#CLM-2265 XTremeGene 9 Sigma-Aldrich Cat#6365779001 Phosphate Buffered Saline, pH 7.4 ThermoFisher Cat#10010031 Critical commercial assays Pierce BCA Protein Assay Kit Thermo Scientific Cat#23227 Pierce Rapid Gold BCA Protein Assay Kit Thermo Scientific Cat#A52335 Trans Blot Turbo RTA Transfer Kit BioRad Cat#1704270 QIAprep Spin Miniprep Kit Qiagen Cat#27106 RNAeasy Mini Kit Qiagen Cat#74104 DNeasy Blood & Tissue Kit Qiagen Cat#69504 QIAmp DNA Blood Maxi Kit Qiagen Cat#51192 Mycoplasma PCR Detection Kit ABM Cat#G238 BioRad Gels 4–20% BioRad Cat#4561096 Deposited data Metabolite Abundance NMDR ST003613 Experimental models: Cell lines Human: A375m Roh-Johnson Lab, University of Utah N/A Human: HCT116 Rabinowitz Lab, Princeton N/A Human: HCT15 Rutter Lab, U of U N/A Human: HEK-293FT Roh-Johnson Lab, U of U N/A Human: LN229 Rabinowitz Lab, Princeton N/A Human: MCF7 Rutter Lab, U of U N/A Human: MDA-MB-468 Roh-Johnson Lab, U of U N/A Human: NCI-H460 McMahon Lab, U of U N/A Human: RPMI7951 ATCC Cat#HTB-66 Human: MeWo Rutter Lab, U of U N/A Human: MM253 Rutter Lab, U of U N/A Human: SKMEL28 Rabinowitz Lab, Princeton N/A Mouse: EO771 Hilgendorf Lab, U of U N/A Experimental models: Organisms/strains NOD.Cg-Rag1tm1Mom Il2rgtm1Wjl/SzJ (NRG) University of Utah Preclinical Research Resource Core N/A Dct-TVA;BrafV600E;Cdkn2alox/lox Grossmann Lab, U of U N/A C57BL6 Charles River Cat#027 (Continued on next page) 16 Cell Reports 44, 115130, January 28, 2025 .. 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 Cat110066 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 ..

    Imaging:

    Article Title: SLC7A5 is required for cancer cell growth under arginine-limited conditions.
    Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER 2-Mercaptoethanol Sigma-Aldrich Cat#M6250 Proteinase K New England Biolabs Cat#P8107S Trypan Blue 0.4% ThermoFisher Cat#15250061 PMSF Cell Signaling Cat#8553S Polybrene Sigma Aldrich Cat#TR-1003 Poly-D-Lysine ThermoFisher Cat#A3890401 Lipofectamine 3000 Transfection Reagent ThermoFisher Cat#L3000008 L-Amino Acid Diet Research Diets Cat#A10021Bi L-Amino Acid Diet -Arginine Research Diets Cat#A10036i qScript cDNA SuperMix VWR Cat#95048 Resazurin sodium salt Sigma-Aldrich Cat#R7017 SYBR Green Master Mix ThermoFisher Cat#A46109 NEBNext Ultra II Q5 Master Mix New England Biolabs Cat#M0544L [1-13C]Citrulline Cambridge Isotopes Cat#CLM-4899 [U-13C]Arginine Cambridge Isotopes Cat#CLM-2265 XTremeGene 9 Sigma-Aldrich Cat#6365779001 Phosphate Buffered Saline, pH 7.4 ThermoFisher Cat#10010031 Critical commercial assays Pierce BCA Protein Assay Kit Thermo Scientific Cat#23227 Pierce Rapid Gold BCA Protein Assay Kit Thermo Scientific Cat#A52335 Trans Blot Turbo RTA Transfer Kit BioRad Cat#1704270 QIAprep Spin Miniprep Kit Qiagen Cat#27106 RNAeasy Mini Kit Qiagen Cat#74104 DNeasy Blood & Tissue Kit Qiagen Cat#69504 QIAmp DNA Blood Maxi Kit Qiagen Cat#51192 Mycoplasma PCR Detection Kit ABM Cat#G238 BioRad Gels 4–20% BioRad Cat#4561096 Deposited data Metabolite Abundance NMDR ST003613 Experimental models: Cell lines Human: A375m Roh-Johnson Lab, University of Utah N/A Human: HCT116 Rabinowitz Lab, Princeton N/A Human: HCT15 Rutter Lab, U of U N/A Human: HEK-293FT Roh-Johnson Lab, U of U N/A Human: LN229 Rabinowitz Lab, Princeton N/A Human: MCF7 Rutter Lab, U of U N/A Human: MDA-MB-468 Roh-Johnson Lab, U of U N/A Human: NCI-H460 McMahon Lab, U of U N/A Human: RPMI7951 ATCC Cat#HTB-66 Human: MeWo Rutter Lab, U of U N/A Human: MM253 Rutter Lab, U of U N/A Human: SKMEL28 Rabinowitz Lab, Princeton N/A Mouse: EO771 Hilgendorf Lab, U of U N/A Experimental models: Organisms/strains NOD.Cg-Rag1tm1Mom Il2rgtm1Wjl/SzJ (NRG) University of Utah Preclinical Research Resource Core N/A Dct-TVA;BrafV600E;Cdkn2alox/lox Grossmann Lab, U of U N/A C57BL6 Charles River Cat#027 (Continued on next page) 16 Cell Reports 44, 115130, January 28, 2025 .. 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 Cat110066 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 ..

    Polymerase Chain Reaction:

    Article Title: SLC7A5 is required for cancer cell growth under arginine-limited conditions.
    Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER 2-Mercaptoethanol Sigma-Aldrich Cat#M6250 Proteinase K New England Biolabs Cat#P8107S Trypan Blue 0.4% ThermoFisher Cat#15250061 PMSF Cell Signaling Cat#8553S Polybrene Sigma Aldrich Cat#TR-1003 Poly-D-Lysine ThermoFisher Cat#A3890401 Lipofectamine 3000 Transfection Reagent ThermoFisher Cat#L3000008 L-Amino Acid Diet Research Diets Cat#A10021Bi L-Amino Acid Diet -Arginine Research Diets Cat#A10036i qScript cDNA SuperMix VWR Cat#95048 Resazurin sodium salt Sigma-Aldrich Cat#R7017 SYBR Green Master Mix ThermoFisher Cat#A46109 NEBNext Ultra II Q5 Master Mix New England Biolabs Cat#M0544L [1-13C]Citrulline Cambridge Isotopes Cat#CLM-4899 [U-13C]Arginine Cambridge Isotopes Cat#CLM-2265 XTremeGene 9 Sigma-Aldrich Cat#6365779001 Phosphate Buffered Saline, pH 7.4 ThermoFisher Cat#10010031 Critical commercial assays Pierce BCA Protein Assay Kit Thermo Scientific Cat#23227 Pierce Rapid Gold BCA Protein Assay Kit Thermo Scientific Cat#A52335 Trans Blot Turbo RTA Transfer Kit BioRad Cat#1704270 QIAprep Spin Miniprep Kit Qiagen Cat#27106 RNAeasy Mini Kit Qiagen Cat#74104 DNeasy Blood & Tissue Kit Qiagen Cat#69504 QIAmp DNA Blood Maxi Kit Qiagen Cat#51192 Mycoplasma PCR Detection Kit ABM Cat#G238 BioRad Gels 4–20% BioRad Cat#4561096 Deposited data Metabolite Abundance NMDR ST003613 Experimental models: Cell lines Human: A375m Roh-Johnson Lab, University of Utah N/A Human: HCT116 Rabinowitz Lab, Princeton N/A Human: HCT15 Rutter Lab, U of U N/A Human: HEK-293FT Roh-Johnson Lab, U of U N/A Human: LN229 Rabinowitz Lab, Princeton N/A Human: MCF7 Rutter Lab, U of U N/A Human: MDA-MB-468 Roh-Johnson Lab, U of U N/A Human: NCI-H460 McMahon Lab, U of U N/A Human: RPMI7951 ATCC Cat#HTB-66 Human: MeWo Rutter Lab, U of U N/A Human: MM253 Rutter Lab, U of U N/A Human: SKMEL28 Rabinowitz Lab, Princeton N/A Mouse: EO771 Hilgendorf Lab, U of U N/A Experimental models: Organisms/strains NOD.Cg-Rag1tm1Mom Il2rgtm1Wjl/SzJ (NRG) University of Utah Preclinical Research Resource Core N/A Dct-TVA;BrafV600E;Cdkn2alox/lox Grossmann Lab, U of U N/A C57BL6 Charles River Cat#027 (Continued on next page) 16 Cell Reports 44, 115130, January 28, 2025 .. 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 Cat110066 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 ..



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    Image Search Results


    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.

    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