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human crispr knockout pooled library brunello  (Addgene inc)


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    Addgene inc human crispr knockout pooled library brunello
    Figure 1. A Pooled Approach for <t>CRISPR</t> Knockout and CRISPRi Screening in Human THP-1 Cells (A) Strategy for preparing CRISPR libraries and performing genetic screens. (B) THP-1-mediated phagocytosis of M. bovis BCG after three rounds of infection (MOI 10:1) with induced green fluorescence (map24::GFP) (Scale bar, 20 mm). (C) Viability of host cells after three rounds of M. bovis BCG infection. (D and E) Expression of Cas9 (D) and dCas9-KRAB (E) in 9 randomly selected monoclonal THP-1 cells. Wild-type THP-1 cells were used as negative control. Vinculin was used as a loading control. (F) An sgRNA for EGFP was introduced in both wild-type and Cas9-expressing THP-1 cells using a lentivirus (pXPR-011) that also contains EGFP as a target (Scale bar, 20 mm). (G) Cas9-expressing THP-1 cells were transduced with an sgRNA targeting AAVS1 at a low MOI. Mutations at the AAVS1 locus were detected by SURVEYOR assay. The size of the AAVS1 amplicon is 500 bp. The cleaved product sizes are 320 and 180 bp. (H) Growth measurement associated with sgRNAs targeting INTS9, MCM2, and non-targeting negative controls sgNC1 and sgNC13. (I and J) RT-qPCR analysis of INTS9 (I) and MCM2 (J) expression in dCas9-KRAB-expressing THP-1 cells. The values are normalized to GAPDH (glyceraldehyde- 3-phosphate dehydrogenase). Data represent the mean ± SD (n = 3) (two-tailed unpaired Student’s t test, *p < 0.05 **p < 0.01 ***p < 0.001). See also Figure S1; Table S13.
    Human Crispr Knockout Pooled Library Brunello, supplied by Addgene inc, used in various techniques. Bioz Stars score: 96/100, based on 277 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/knockout+library/Human+CRISPR+Knockout+Pooled+Library+(Brunello)+(Pooled+Library+%2373179%2C+%2373179-LV%2C+%2373178%2C+%2373178-LV)/pm32970993-253-0-9
    Average 96 stars, based on 277 article reviews
    human crispr knockout pooled library brunello - by Bioz Stars, 2026-09
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    Images

    1) Product Images from "Illuminating Host-Mycobacterial Interactions with Genome-wide CRISPR Knockout and CRISPRi Screens."

    Article Title: Illuminating Host-Mycobacterial Interactions with Genome-wide CRISPR Knockout and CRISPRi Screens.

    Journal: Cell systems

    doi: 10.1016/j.cels.2020.08.010

    Figure 1. A Pooled Approach for CRISPR Knockout and CRISPRi Screening in Human THP-1 Cells (A) Strategy for preparing CRISPR libraries and performing genetic screens. (B) THP-1-mediated phagocytosis of M. bovis BCG after three rounds of infection (MOI 10:1) with induced green fluorescence (map24::GFP) (Scale bar, 20 mm). (C) Viability of host cells after three rounds of M. bovis BCG infection. (D and E) Expression of Cas9 (D) and dCas9-KRAB (E) in 9 randomly selected monoclonal THP-1 cells. Wild-type THP-1 cells were used as negative control. Vinculin was used as a loading control. (F) An sgRNA for EGFP was introduced in both wild-type and Cas9-expressing THP-1 cells using a lentivirus (pXPR-011) that also contains EGFP as a target (Scale bar, 20 mm). (G) Cas9-expressing THP-1 cells were transduced with an sgRNA targeting AAVS1 at a low MOI. Mutations at the AAVS1 locus were detected by SURVEYOR assay. The size of the AAVS1 amplicon is 500 bp. The cleaved product sizes are 320 and 180 bp. (H) Growth measurement associated with sgRNAs targeting INTS9, MCM2, and non-targeting negative controls sgNC1 and sgNC13. (I and J) RT-qPCR analysis of INTS9 (I) and MCM2 (J) expression in dCas9-KRAB-expressing THP-1 cells. The values are normalized to GAPDH (glyceraldehyde- 3-phosphate dehydrogenase). Data represent the mean ± SD (n = 3) (two-tailed unpaired Student’s t test, *p < 0.05 **p < 0.01 ***p < 0.001). See also Figure S1; Table S13.
    Figure Legend Snippet: Figure 1. A Pooled Approach for CRISPR Knockout and CRISPRi Screening in Human THP-1 Cells (A) Strategy for preparing CRISPR libraries and performing genetic screens. (B) THP-1-mediated phagocytosis of M. bovis BCG after three rounds of infection (MOI 10:1) with induced green fluorescence (map24::GFP) (Scale bar, 20 mm). (C) Viability of host cells after three rounds of M. bovis BCG infection. (D and E) Expression of Cas9 (D) and dCas9-KRAB (E) in 9 randomly selected monoclonal THP-1 cells. Wild-type THP-1 cells were used as negative control. Vinculin was used as a loading control. (F) An sgRNA for EGFP was introduced in both wild-type and Cas9-expressing THP-1 cells using a lentivirus (pXPR-011) that also contains EGFP as a target (Scale bar, 20 mm). (G) Cas9-expressing THP-1 cells were transduced with an sgRNA targeting AAVS1 at a low MOI. Mutations at the AAVS1 locus were detected by SURVEYOR assay. The size of the AAVS1 amplicon is 500 bp. The cleaved product sizes are 320 and 180 bp. (H) Growth measurement associated with sgRNAs targeting INTS9, MCM2, and non-targeting negative controls sgNC1 and sgNC13. (I and J) RT-qPCR analysis of INTS9 (I) and MCM2 (J) expression in dCas9-KRAB-expressing THP-1 cells. The values are normalized to GAPDH (glyceraldehyde- 3-phosphate dehydrogenase). Data represent the mean ± SD (n = 3) (two-tailed unpaired Student’s t test, *p < 0.05 **p < 0.01 ***p < 0.001). See also Figure S1; Table S13.

    Techniques Used: CRISPR, Knock-Out, Infection, Expressing, Negative Control, Control, Transduction, Amplification, Quantitative RT-PCR, Two Tailed Test

    Figure 2. Genome-wide Pooled CRISPR Knockout and CRISPRi Screens to Dissect Biological Pathways in Mycobacterial Infection (A and B) Volcano plots from CRISPR knockout (A) and CRISPRi (B) screens. For each sgRNA-targeted gene, the x axis shows its enrichment or depletion post- infection, and the y axis shows statistical significance measured by p value. Positive and negative screen hits are labeled as red and green dots, respectively. Gray dots represent non-targeting controls. For each screen, experiments were carried out in triplicate. (C) Enriched genes in the Venn diagram were filtered with a cut-off of FDR <0.1 and log2-fold change >1 in M. bovis BCG infection. The degree of significance of the overlap is given. (D) Gene-centric visualization of average fold change of CRISPR knockout and CRISPRi screens in infected versus non-infected host cells. Selected type I IFN and AHR/ARNT pathway components are highlighted in orange and blue. (E and F) Candidate genes identified by CRISPR knockout (E) and CRISPRi (F) screens were functionally categorized to understand the changes in biological functions involved in M. bovis BCG infection. Pathways shown in red are those identified by both screens. Color gradient of nodes represents the enrichment scores of gene sets. Node size represents the number of genes in the gene set. Edge width represents mutual overlap of genes. See also Figures S2 and S3; Tables S1, S2, S3, S4, S5, S11, and S12.
    Figure Legend Snippet: Figure 2. Genome-wide Pooled CRISPR Knockout and CRISPRi Screens to Dissect Biological Pathways in Mycobacterial Infection (A and B) Volcano plots from CRISPR knockout (A) and CRISPRi (B) screens. For each sgRNA-targeted gene, the x axis shows its enrichment or depletion post- infection, and the y axis shows statistical significance measured by p value. Positive and negative screen hits are labeled as red and green dots, respectively. Gray dots represent non-targeting controls. For each screen, experiments were carried out in triplicate. (C) Enriched genes in the Venn diagram were filtered with a cut-off of FDR <0.1 and log2-fold change >1 in M. bovis BCG infection. The degree of significance of the overlap is given. (D) Gene-centric visualization of average fold change of CRISPR knockout and CRISPRi screens in infected versus non-infected host cells. Selected type I IFN and AHR/ARNT pathway components are highlighted in orange and blue. (E and F) Candidate genes identified by CRISPR knockout (E) and CRISPRi (F) screens were functionally categorized to understand the changes in biological functions involved in M. bovis BCG infection. Pathways shown in red are those identified by both screens. Color gradient of nodes represents the enrichment scores of gene sets. Node size represents the number of genes in the gene set. Edge width represents mutual overlap of genes. See also Figures S2 and S3; Tables S1, S2, S3, S4, S5, S11, and S12.

    Techniques Used: Genome Wide, CRISPR, Knock-Out, Infection, Labeling

    Figure 3. Secondary CRISPR Knockout and CRISPRi Screens Identify Host Genetic Hits in Mycobacterial Infection (A) Enriched genes were filtered with a cut-off of FDR <0.05 and log2-fold change >0.5 in M. bovis BCG infection. The degree of significance of the overlap is given. (B) Validation rate of genetic hits in secondary screens grouped by their p values in primary genome-wide screens in M. bovis BCG infection. Number of genes per category is indicated. (C) Genetic hits from both primary and secondary screens were ranked by their differential sgRNA abundance between M. bovis BCG-infected versus uninfected populations (log2 fold change). (D) Heatmap of screen hits (log2 fold change) clustered in different biological pathways in M. bovis BCG infection. See also Figures S4 and S5; Tables S6, S7, S8, S9, and S10.
    Figure Legend Snippet: Figure 3. Secondary CRISPR Knockout and CRISPRi Screens Identify Host Genetic Hits in Mycobacterial Infection (A) Enriched genes were filtered with a cut-off of FDR <0.05 and log2-fold change >0.5 in M. bovis BCG infection. The degree of significance of the overlap is given. (B) Validation rate of genetic hits in secondary screens grouped by their p values in primary genome-wide screens in M. bovis BCG infection. Number of genes per category is indicated. (C) Genetic hits from both primary and secondary screens were ranked by their differential sgRNA abundance between M. bovis BCG-infected versus uninfected populations (log2 fold change). (D) Heatmap of screen hits (log2 fold change) clustered in different biological pathways in M. bovis BCG infection. See also Figures S4 and S5; Tables S6, S7, S8, S9, and S10.

    Techniques Used: CRISPR, Knock-Out, Infection, Biomarker Discovery, Genome Wide

    Related Articles

    Transduction:

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    Article Snippet: .. WT HAP1, RNF25 KO and ZNF598 KO HAP1 cells were transduced with the pre-packaged genome-wide All-in-One Brunello lentiviral library (Addgene 73179) at a multiplicity of infection (MOI) of 0.25 and at 500× representation. ..

    Genome Wide:

    Article Title: RNF25 confers mRNA damage tolerance by curbing activation of the integrated stress response.
    Article Snippet: .. WT HAP1, RNF25 KO and ZNF598 KO HAP1 cells were transduced with the pre-packaged genome-wide All-in-One Brunello lentiviral library (Addgene 73179) at a multiplicity of infection (MOI) of 0.25 and at 500× representation. ..

    Article Title: RNF25 confers mRNA damage tolerance by curbing activation of the integrated stress response.
    Article Snippet: MS-5 mouse cells DSMZ ACC 441 .. Oligonucleotides Oligonucleotide sequences used in this study are provided in Table S9 This study N/A Recombinant DNA pcDNA5/FRT/TO-RNF25-WT-mVenus3XFlag This study N/A pcDNA5/FRT/TO-RNF25-ΔRWD-mVenus3XFlag This study N/A pcDNA5/FRT/TO-RNF25-ΔRING-mVenus3XFlag This study N/A pcDNA5/FRT/TO-mVenus-3xFlagGateway Di Fiore and Pines80 Addgene Plasmid #40999 pOG44_FLP_recombinase Hallacli et al.81 Addgene Plasmid #209087 Genome-wide All-in-One Brunello lentiviral library Doench et al.82 Addgene pooled library #73179 pX330-Puro Pritchard et al.83 Addgene Plasmid #82580 pU6-tevopreq1-GG-acceptor vector Nelson et al.84 Addgene Plasmid #174038 pU6-pegRNA-GG-acceptor vector Anzalone et al.85 Addgene Plasmid #132777 pCMV-PEmax Chen et al.86 Addgene Plasmid #174820 pEF1a-hMLH1dn Chen et al.86 Addgene Plasmid #174824 lentiGuide-Puro Sanjana et al.87 Addgene Plasmid #52963 pMDLg/pRRE Dull et al.88 Addgene Plasmid #12251 pRSV-Rev Dull et al.88 Addgene Plasmid #12253 pMD2.G/pVSV-g Didier Trono Addgene Plasmid #12259 Software and algorithms ImageLab (Version 5.2) Bio-Rad RRID:SCR_014210 SparkControl 3.1 Tecan https://lifesciences.tecan.com/multimode- plate-reader?p=tab–3 R (version 4.3.2) R Project for Statistical Computing RRID:SCR_001905 GraphPad Prism (Version 9.1.0) Dotmatics RRID:SCR_002798 R package limma (v. 3.58.1) Bioconductor RRID:SCR_010943 MaxQuant (v.1.5.2.8) MaxQuant RRID:SCR_014485 PhosphoSitePlus Kinase Library Johnson et al.61 https://kinase-library.phosphosite.org Snapgene (Version 5.0.8) GSL Biotech LLC RRID:SCR_015052 R package preprocessCore (Version 1.64.0) Bioconductor https://bioconductor.org/packages/ release/bioc/html/preprocessCore.html cutadapt (version see respective methods section) Martin89 https://cutadapt.readthedocs.io; RRID:SCR_011841 Bowtie2 (version 2.5.0) Langmead and Salzberg90 http://bowtie-bio.sourceforge.net/bowtie2/ index.shtml; RRID:SCR_016368 Samtools Li et al.91 http://samtools.sourceforge.net/; RRID:SCR_002105 STAR (version see respective methods section) Dobin et al.92 https://github.com/alexdobin/STAR; RRID:SCR_004463 Scikit-ribo, modified version Fang et al.93 Gao et al.94 https://github.com/nedialkova-lab/scikitribo-ext (Continued on next page) e4 Molecular Cell 86, 1275–1292.e1–e12, April 2, 2026 ..

    Infection:

    Article Title: RNF25 confers mRNA damage tolerance by curbing activation of the integrated stress response.
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    CRISPR:

    Article Title: ULK1 drives NDP52-mediated selective autophagic degradation of MHC-I to promote immune evasion in HPV-positive head and neck cancer
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    Article Title: ADA2-deficient cells exhibit increased levels of cell death and metabolic disturbances
    Article Snippet: .. Single-guide RNAs targeting ADA2 from the human CRISPR Brunello library (#73179; addgene, Watertown, MA, USA) [ ] were cloned into the lentiCRISPRv2 puro plasmid. lentiCRISPRv2 puro was a gift from Brett Stringer (plasmid #98290; addgene; http://n2t.net/addgene:98290 ; RRID:Addgene_98290). .. U-937 cells and Jurkat cells were transfected by electroporation using the NeonTM Transfection System (#MPK5000; Thermo Fisher Scientific) according to the manufacturer’s instructions [ ].

    Article Title: Genome-wide CRISPR screens identify the EXO1-CAF-1 pathway suppressing R-loop-associated DNA damage.
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    Article Title: ADA2-deficient cells exhibit increased levels of cell death and metabolic disturbances.
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    Article Title: Genome-wide CRISPR screens identify the EXO1-CAF-1 pathway suppressing R-loop-associated DNA damage
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    Article Title: ZAP targets aberrant mRNA transcripts encoding proteins with defective signal peptides for degradation.
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    Clone Assay:

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    Article Title: ADA2-deficient cells exhibit increased levels of cell death and metabolic disturbances.
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    Plasmid Preparation:

    Article Title: ADA2-deficient cells exhibit increased levels of cell death and metabolic disturbances
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    Article Title: RNF25 confers mRNA damage tolerance by curbing activation of the integrated stress response.
    Article Snippet: MS-5 mouse cells DSMZ ACC 441 .. Oligonucleotides Oligonucleotide sequences used in this study are provided in Table S9 This study N/A Recombinant DNA pcDNA5/FRT/TO-RNF25-WT-mVenus3XFlag This study N/A pcDNA5/FRT/TO-RNF25-ΔRWD-mVenus3XFlag This study N/A pcDNA5/FRT/TO-RNF25-ΔRING-mVenus3XFlag This study N/A pcDNA5/FRT/TO-mVenus-3xFlagGateway Di Fiore and Pines80 Addgene Plasmid #40999 pOG44_FLP_recombinase Hallacli et al.81 Addgene Plasmid #209087 Genome-wide All-in-One Brunello lentiviral library Doench et al.82 Addgene pooled library #73179 pX330-Puro Pritchard et al.83 Addgene Plasmid #82580 pU6-tevopreq1-GG-acceptor vector Nelson et al.84 Addgene Plasmid #174038 pU6-pegRNA-GG-acceptor vector Anzalone et al.85 Addgene Plasmid #132777 pCMV-PEmax Chen et al.86 Addgene Plasmid #174820 pEF1a-hMLH1dn Chen et al.86 Addgene Plasmid #174824 lentiGuide-Puro Sanjana et al.87 Addgene Plasmid #52963 pMDLg/pRRE Dull et al.88 Addgene Plasmid #12251 pRSV-Rev Dull et al.88 Addgene Plasmid #12253 pMD2.G/pVSV-g Didier Trono Addgene Plasmid #12259 Software and algorithms ImageLab (Version 5.2) Bio-Rad RRID:SCR_014210 SparkControl 3.1 Tecan https://lifesciences.tecan.com/multimode- plate-reader?p=tab–3 R (version 4.3.2) R Project for Statistical Computing RRID:SCR_001905 GraphPad Prism (Version 9.1.0) Dotmatics RRID:SCR_002798 R package limma (v. 3.58.1) Bioconductor RRID:SCR_010943 MaxQuant (v.1.5.2.8) MaxQuant RRID:SCR_014485 PhosphoSitePlus Kinase Library Johnson et al.61 https://kinase-library.phosphosite.org Snapgene (Version 5.0.8) GSL Biotech LLC RRID:SCR_015052 R package preprocessCore (Version 1.64.0) Bioconductor https://bioconductor.org/packages/ release/bioc/html/preprocessCore.html cutadapt (version see respective methods section) Martin89 https://cutadapt.readthedocs.io; RRID:SCR_011841 Bowtie2 (version 2.5.0) Langmead and Salzberg90 http://bowtie-bio.sourceforge.net/bowtie2/ index.shtml; RRID:SCR_016368 Samtools Li et al.91 http://samtools.sourceforge.net/; RRID:SCR_002105 STAR (version see respective methods section) Dobin et al.92 https://github.com/alexdobin/STAR; RRID:SCR_004463 Scikit-ribo, modified version Fang et al.93 Gao et al.94 https://github.com/nedialkova-lab/scikitribo-ext (Continued on next page) e4 Molecular Cell 86, 1275–1292.e1–e12, April 2, 2026 ..

    Article Title: ADA2-deficient cells exhibit increased levels of cell death and metabolic disturbances.
    Article Snippet: .. Generation of ADA2-/- cell lines by CRISPR/Cas9 Single-guide RNAs targeting ADA2 from the human CRISPR Brunello library (#73179; addgene, Watertown, MA, USA)34 were cloned into the lentiCRISPRv2 puro plasmid. lentiCRISPRv2 puro was a gift from Brett Stringer (plasmid #98290; addgene; http://n2t.net/addgene:98290; RRID:Addgene_98290). .. U-937 cells and Jurkat cells were transfected by electroporation using the AR TI CL E IN P RE SS NeonTM Transfection System (#MPK5000; Thermo Fisher Scientific) according to the manufacturer’s instructions.33

    Recombinant:

    Article Title: RNF25 confers mRNA damage tolerance by curbing activation of the integrated stress response.
    Article Snippet: MS-5 mouse cells DSMZ ACC 441 .. Oligonucleotides Oligonucleotide sequences used in this study are provided in Table S9 This study N/A Recombinant DNA pcDNA5/FRT/TO-RNF25-WT-mVenus3XFlag This study N/A pcDNA5/FRT/TO-RNF25-ΔRWD-mVenus3XFlag This study N/A pcDNA5/FRT/TO-RNF25-ΔRING-mVenus3XFlag This study N/A pcDNA5/FRT/TO-mVenus-3xFlagGateway Di Fiore and Pines80 Addgene Plasmid #40999 pOG44_FLP_recombinase Hallacli et al.81 Addgene Plasmid #209087 Genome-wide All-in-One Brunello lentiviral library Doench et al.82 Addgene pooled library #73179 pX330-Puro Pritchard et al.83 Addgene Plasmid #82580 pU6-tevopreq1-GG-acceptor vector Nelson et al.84 Addgene Plasmid #174038 pU6-pegRNA-GG-acceptor vector Anzalone et al.85 Addgene Plasmid #132777 pCMV-PEmax Chen et al.86 Addgene Plasmid #174820 pEF1a-hMLH1dn Chen et al.86 Addgene Plasmid #174824 lentiGuide-Puro Sanjana et al.87 Addgene Plasmid #52963 pMDLg/pRRE Dull et al.88 Addgene Plasmid #12251 pRSV-Rev Dull et al.88 Addgene Plasmid #12253 pMD2.G/pVSV-g Didier Trono Addgene Plasmid #12259 Software and algorithms ImageLab (Version 5.2) Bio-Rad RRID:SCR_014210 SparkControl 3.1 Tecan https://lifesciences.tecan.com/multimode- plate-reader?p=tab–3 R (version 4.3.2) R Project for Statistical Computing RRID:SCR_001905 GraphPad Prism (Version 9.1.0) Dotmatics RRID:SCR_002798 R package limma (v. 3.58.1) Bioconductor RRID:SCR_010943 MaxQuant (v.1.5.2.8) MaxQuant RRID:SCR_014485 PhosphoSitePlus Kinase Library Johnson et al.61 https://kinase-library.phosphosite.org Snapgene (Version 5.0.8) GSL Biotech LLC RRID:SCR_015052 R package preprocessCore (Version 1.64.0) Bioconductor https://bioconductor.org/packages/ release/bioc/html/preprocessCore.html cutadapt (version see respective methods section) Martin89 https://cutadapt.readthedocs.io; RRID:SCR_011841 Bowtie2 (version 2.5.0) Langmead and Salzberg90 http://bowtie-bio.sourceforge.net/bowtie2/ index.shtml; RRID:SCR_016368 Samtools Li et al.91 http://samtools.sourceforge.net/; RRID:SCR_002105 STAR (version see respective methods section) Dobin et al.92 https://github.com/alexdobin/STAR; RRID:SCR_004463 Scikit-ribo, modified version Fang et al.93 Gao et al.94 https://github.com/nedialkova-lab/scikitribo-ext (Continued on next page) e4 Molecular Cell 86, 1275–1292.e1–e12, April 2, 2026 ..

    Software:

    Article Title: RNF25 confers mRNA damage tolerance by curbing activation of the integrated stress response.
    Article Snippet: MS-5 mouse cells DSMZ ACC 441 .. Oligonucleotides Oligonucleotide sequences used in this study are provided in Table S9 This study N/A Recombinant DNA pcDNA5/FRT/TO-RNF25-WT-mVenus3XFlag This study N/A pcDNA5/FRT/TO-RNF25-ΔRWD-mVenus3XFlag This study N/A pcDNA5/FRT/TO-RNF25-ΔRING-mVenus3XFlag This study N/A pcDNA5/FRT/TO-mVenus-3xFlagGateway Di Fiore and Pines80 Addgene Plasmid #40999 pOG44_FLP_recombinase Hallacli et al.81 Addgene Plasmid #209087 Genome-wide All-in-One Brunello lentiviral library Doench et al.82 Addgene pooled library #73179 pX330-Puro Pritchard et al.83 Addgene Plasmid #82580 pU6-tevopreq1-GG-acceptor vector Nelson et al.84 Addgene Plasmid #174038 pU6-pegRNA-GG-acceptor vector Anzalone et al.85 Addgene Plasmid #132777 pCMV-PEmax Chen et al.86 Addgene Plasmid #174820 pEF1a-hMLH1dn Chen et al.86 Addgene Plasmid #174824 lentiGuide-Puro Sanjana et al.87 Addgene Plasmid #52963 pMDLg/pRRE Dull et al.88 Addgene Plasmid #12251 pRSV-Rev Dull et al.88 Addgene Plasmid #12253 pMD2.G/pVSV-g Didier Trono Addgene Plasmid #12259 Software and algorithms ImageLab (Version 5.2) Bio-Rad RRID:SCR_014210 SparkControl 3.1 Tecan https://lifesciences.tecan.com/multimode- plate-reader?p=tab–3 R (version 4.3.2) R Project for Statistical Computing RRID:SCR_001905 GraphPad Prism (Version 9.1.0) Dotmatics RRID:SCR_002798 R package limma (v. 3.58.1) Bioconductor RRID:SCR_010943 MaxQuant (v.1.5.2.8) MaxQuant RRID:SCR_014485 PhosphoSitePlus Kinase Library Johnson et al.61 https://kinase-library.phosphosite.org Snapgene (Version 5.0.8) GSL Biotech LLC RRID:SCR_015052 R package preprocessCore (Version 1.64.0) Bioconductor https://bioconductor.org/packages/ release/bioc/html/preprocessCore.html cutadapt (version see respective methods section) Martin89 https://cutadapt.readthedocs.io; RRID:SCR_011841 Bowtie2 (version 2.5.0) Langmead and Salzberg90 http://bowtie-bio.sourceforge.net/bowtie2/ index.shtml; RRID:SCR_016368 Samtools Li et al.91 http://samtools.sourceforge.net/; RRID:SCR_002105 STAR (version see respective methods section) Dobin et al.92 https://github.com/alexdobin/STAR; RRID:SCR_004463 Scikit-ribo, modified version Fang et al.93 Gao et al.94 https://github.com/nedialkova-lab/scikitribo-ext (Continued on next page) e4 Molecular Cell 86, 1275–1292.e1–e12, April 2, 2026 ..

    Modification:

    Article Title: RNF25 confers mRNA damage tolerance by curbing activation of the integrated stress response.
    Article Snippet: MS-5 mouse cells DSMZ ACC 441 .. Oligonucleotides Oligonucleotide sequences used in this study are provided in Table S9 This study N/A Recombinant DNA pcDNA5/FRT/TO-RNF25-WT-mVenus3XFlag This study N/A pcDNA5/FRT/TO-RNF25-ΔRWD-mVenus3XFlag This study N/A pcDNA5/FRT/TO-RNF25-ΔRING-mVenus3XFlag This study N/A pcDNA5/FRT/TO-mVenus-3xFlagGateway Di Fiore and Pines80 Addgene Plasmid #40999 pOG44_FLP_recombinase Hallacli et al.81 Addgene Plasmid #209087 Genome-wide All-in-One Brunello lentiviral library Doench et al.82 Addgene pooled library #73179 pX330-Puro Pritchard et al.83 Addgene Plasmid #82580 pU6-tevopreq1-GG-acceptor vector Nelson et al.84 Addgene Plasmid #174038 pU6-pegRNA-GG-acceptor vector Anzalone et al.85 Addgene Plasmid #132777 pCMV-PEmax Chen et al.86 Addgene Plasmid #174820 pEF1a-hMLH1dn Chen et al.86 Addgene Plasmid #174824 lentiGuide-Puro Sanjana et al.87 Addgene Plasmid #52963 pMDLg/pRRE Dull et al.88 Addgene Plasmid #12251 pRSV-Rev Dull et al.88 Addgene Plasmid #12253 pMD2.G/pVSV-g Didier Trono Addgene Plasmid #12259 Software and algorithms ImageLab (Version 5.2) Bio-Rad RRID:SCR_014210 SparkControl 3.1 Tecan https://lifesciences.tecan.com/multimode- plate-reader?p=tab–3 R (version 4.3.2) R Project for Statistical Computing RRID:SCR_001905 GraphPad Prism (Version 9.1.0) Dotmatics RRID:SCR_002798 R package limma (v. 3.58.1) Bioconductor RRID:SCR_010943 MaxQuant (v.1.5.2.8) MaxQuant RRID:SCR_014485 PhosphoSitePlus Kinase Library Johnson et al.61 https://kinase-library.phosphosite.org Snapgene (Version 5.0.8) GSL Biotech LLC RRID:SCR_015052 R package preprocessCore (Version 1.64.0) Bioconductor https://bioconductor.org/packages/ release/bioc/html/preprocessCore.html cutadapt (version see respective methods section) Martin89 https://cutadapt.readthedocs.io; RRID:SCR_011841 Bowtie2 (version 2.5.0) Langmead and Salzberg90 http://bowtie-bio.sourceforge.net/bowtie2/ index.shtml; RRID:SCR_016368 Samtools Li et al.91 http://samtools.sourceforge.net/; RRID:SCR_002105 STAR (version see respective methods section) Dobin et al.92 https://github.com/alexdobin/STAR; RRID:SCR_004463 Scikit-ribo, modified version Fang et al.93 Gao et al.94 https://github.com/nedialkova-lab/scikitribo-ext (Continued on next page) e4 Molecular Cell 86, 1275–1292.e1–e12, April 2, 2026 ..

    Knock-Out:

    Article Title: Genome-wide CRISPR screens identify the EXO1-CAF-1 pathway suppressing R-loop-associated DNA damage.
    Article Snippet: Antibodies used for western blot, at 1:500 dilution, were: EXO1 (Novus NBP2-16391); CHAF1A (Cell Signaling Technology 5480s); RNAseH1 (Invitrogen P A5-P A598119); Vinculin (Santa Cruz Biotechnology sc-73614): GAPDH (Santa Cruz Biotechnology sc-47724). .. For CRISPR knockout screens, the Brunello Human CRISPR knockout pooled lentiviral library (Addgene 73179) was used [ 43 ]. .. Antibodies used for western blot, at 1:500 dilution, were: EXO1 (Novus NBP2-16391); CHAF1A (Cell Signaling Technology 5480s); RNAseH1 (Invitrogen P A5-P A598119); Vinculin (Santa Cruz Biotechnology sc-73614): GAPDH (Santa Cruz Biotechnology sc-47724).

    Article Title: Genome-wide CRISPR screens identify the EXO1-CAF-1 pathway suppressing R-loop-associated DNA damage
    Article Snippet: Antibodies used for western blot, at 1:500 dilution, were: EXO1 (Novus NBP2-16391); CHAF1A (Cell Signaling Technology 5480s); RNAseH1 (Invitrogen PA5- PA598119 ); Vinculin (Santa Cruz Biotechnology sc-73614): GAPDH (Santa Cruz Biotechnology sc-47724). .. For CRISPR knockout screens, the Brunello Human CRISPR knockout pooled lentiviral library (Addgene 73179) was used [ ]. ..

    Article Title: ZAP targets aberrant mRNA transcripts encoding proteins with defective signal peptides for degradation.
    Article Snippet: Band intensities were quantified with ImageJ 1.53 K. Primary antibodies used: FLAG (Sigma-Aldrich, A8592), GFP (Clontech, 632381), ZAP (Proteintech, 16820-1-AP), β-actin (Cell Signaling Technology, 51255), HA (Sigma-Aldrich, 12013819001), eIF2α (Cell Signaling Technology, 9722), phospho-eIF2α (Abcam, ab32157), SRP54 (BD Biosciences, 610940), vinculin (Santa Cruz Biotechnology, sc-73614), SRPRB (Bethyl Laboratory, A305-440A), VDAC (Cell Signaling Technology, 4866), calnexin (Enzo, ADI-SPA-860), calreticulin (Novus Biologicals, NB600-101), and PERK (Cell Signaling Technology, 3192). .. Flow cytometric CRISPR screen Lentivirus was generated from the Brunello human CRISPR knockout pooled library (Sanson et al, 2018) (Addgene, #73179) as described before (Coria et al, 2025). .. To ensure proper Cas9 expression, RAPP reporter cells (PPL-WT and PPL-Δ2 L) were kept under 10 μg/mL of blasticidin (InvivoGen, ant-bl-05) until a day before lentiviral transduction.

    Generated:

    Article Title: ZAP targets aberrant mRNA transcripts encoding proteins with defective signal peptides for degradation.
    Article Snippet: Band intensities were quantified with ImageJ 1.53 K. Primary antibodies used: FLAG (Sigma-Aldrich, A8592), GFP (Clontech, 632381), ZAP (Proteintech, 16820-1-AP), β-actin (Cell Signaling Technology, 51255), HA (Sigma-Aldrich, 12013819001), eIF2α (Cell Signaling Technology, 9722), phospho-eIF2α (Abcam, ab32157), SRP54 (BD Biosciences, 610940), vinculin (Santa Cruz Biotechnology, sc-73614), SRPRB (Bethyl Laboratory, A305-440A), VDAC (Cell Signaling Technology, 4866), calnexin (Enzo, ADI-SPA-860), calreticulin (Novus Biologicals, NB600-101), and PERK (Cell Signaling Technology, 3192). .. Flow cytometric CRISPR screen Lentivirus was generated from the Brunello human CRISPR knockout pooled library (Sanson et al, 2018) (Addgene, #73179) as described before (Coria et al, 2025). .. To ensure proper Cas9 expression, RAPP reporter cells (PPL-WT and PPL-Δ2 L) were kept under 10 μg/mL of blasticidin (InvivoGen, ant-bl-05) until a day before lentiviral transduction.



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    Image Search Results


    Genome-wide CRISPR knockout screens for proliferation and cisplatin sensitization of HeLa WT and EXO1-knockout cells. ( A ) Overview of the CRISPR knockout screens to identify genes that are required for proliferation and cisplatin sensitivity of WT and EXO1-knockout HeLa cells. Created in BioRender. Moldovan, G. (2026); https://BioRender.com/c77sziu . ( B ) The cellular survival of WT and EXO1-knockout HeLa cells at each splitting time. Survival was calculated by dividing the number of live cells in the cisplatin-treatment population to the control (no treatment) population.

    Journal: Nucleic Acids Research

    Article Title: Genome-wide CRISPR screens identify the EXO1-CAF-1 pathway suppressing R-loop-associated DNA damage

    doi: 10.1093/nar/gkag226

    Figure Lengend Snippet: Genome-wide CRISPR knockout screens for proliferation and cisplatin sensitization of HeLa WT and EXO1-knockout cells. ( A ) Overview of the CRISPR knockout screens to identify genes that are required for proliferation and cisplatin sensitivity of WT and EXO1-knockout HeLa cells. Created in BioRender. Moldovan, G. (2026); https://BioRender.com/c77sziu . ( B ) The cellular survival of WT and EXO1-knockout HeLa cells at each splitting time. Survival was calculated by dividing the number of live cells in the cisplatin-treatment population to the control (no treatment) population.

    Article Snippet: For CRISPR knockout screens, the Brunello Human CRISPR knockout pooled lentiviral library (Addgene 73179) was used [ ].

    Techniques: Genome Wide, CRISPR, Knock-Out, Control

    Analyses of the cisplatin sensitivity CRISPR screens in WT and EXO1-knockout cells. ( A ) Functional annotation clustering of the top hits with MAGeCK score lower than 0.005 which cause cisplatin sensitivity in WT HeLa cells, using Gene Ontology and Uniprot terms. ( B ) Table showing the biological processes and corresponding genes from the Gene Ontology pathway analysis of the top hits with MAGeCK score lower than 0.005 which cause cisplatin sensitivity in WT HeLa cells. GO_BP terms with negative logP >1 are presented. ( C, D ) Functional annotation clustering of the top hits with MAGeCK score lower than 0.005 which cause cisplatin sensitivity in HeLa-EXO1 KO#1 ( C ) and HeLa-EXO1 KO#3 ( D ) cells using Gene Ontology and Uniprot terms. ( E ) Diagram showing the overlap of identical genes within the top cisplatin sensitivity hits in the two EXO1-knockout cell lines. The number of genes with MAGeCK score lower than 0.005 (left) and 0.015 (right) are shown. ( F ) The number of common genes within the top cisplatin sensitivity hits in the two EXO1-knockout cell lines compared to the random probability of identical hits. The top hits with MAGeCK score lower than 0.005 (left) and 0.015 (right) were included in the analysis. ( G ) Diagram showing the overlap of identical genes within the top cisplatin sensitivity hits in WT and EXO1 KO#1 cells. The number of genes with MAGeCK score lower than 0.005 (left) and 0.015 (right) are shown. ( H ) The number of common genes within the top cisplatin sensitivity hits in WT and EXO1 KO#1 cells compared to the random probability of identical hits. The top hits with MAGeCK score lower than 0.005 (left) and 0.015 (right) were included in the analysis. ( I ) Diagram showing the overlap of identical genes within the top cisplatin sensitivity hits in WT and EXO1 KO#3 cells. The number of genes with MAGeCK score lower than 0.005 (left) and 0.015 (right) are shown. ( J ) The number of common genes within the top cisplatin sensitivity hits in WT and EXO1 KO#3 cells compared to the random probability of identical hits. The top hits with MAGeCK score lower than 0.005 (left) and 0.015 (right) were included in the analysis.

    Journal: Nucleic Acids Research

    Article Title: Genome-wide CRISPR screens identify the EXO1-CAF-1 pathway suppressing R-loop-associated DNA damage

    doi: 10.1093/nar/gkag226

    Figure Lengend Snippet: Analyses of the cisplatin sensitivity CRISPR screens in WT and EXO1-knockout cells. ( A ) Functional annotation clustering of the top hits with MAGeCK score lower than 0.005 which cause cisplatin sensitivity in WT HeLa cells, using Gene Ontology and Uniprot terms. ( B ) Table showing the biological processes and corresponding genes from the Gene Ontology pathway analysis of the top hits with MAGeCK score lower than 0.005 which cause cisplatin sensitivity in WT HeLa cells. GO_BP terms with negative logP >1 are presented. ( C, D ) Functional annotation clustering of the top hits with MAGeCK score lower than 0.005 which cause cisplatin sensitivity in HeLa-EXO1 KO#1 ( C ) and HeLa-EXO1 KO#3 ( D ) cells using Gene Ontology and Uniprot terms. ( E ) Diagram showing the overlap of identical genes within the top cisplatin sensitivity hits in the two EXO1-knockout cell lines. The number of genes with MAGeCK score lower than 0.005 (left) and 0.015 (right) are shown. ( F ) The number of common genes within the top cisplatin sensitivity hits in the two EXO1-knockout cell lines compared to the random probability of identical hits. The top hits with MAGeCK score lower than 0.005 (left) and 0.015 (right) were included in the analysis. ( G ) Diagram showing the overlap of identical genes within the top cisplatin sensitivity hits in WT and EXO1 KO#1 cells. The number of genes with MAGeCK score lower than 0.005 (left) and 0.015 (right) are shown. ( H ) The number of common genes within the top cisplatin sensitivity hits in WT and EXO1 KO#1 cells compared to the random probability of identical hits. The top hits with MAGeCK score lower than 0.005 (left) and 0.015 (right) were included in the analysis. ( I ) Diagram showing the overlap of identical genes within the top cisplatin sensitivity hits in WT and EXO1 KO#3 cells. The number of genes with MAGeCK score lower than 0.005 (left) and 0.015 (right) are shown. ( J ) The number of common genes within the top cisplatin sensitivity hits in WT and EXO1 KO#3 cells compared to the random probability of identical hits. The top hits with MAGeCK score lower than 0.005 (left) and 0.015 (right) were included in the analysis.

    Article Snippet: For CRISPR knockout screens, the Brunello Human CRISPR knockout pooled lentiviral library (Addgene 73179) was used [ ].

    Techniques: CRISPR, Knock-Out, Functional Assay

    Analyses of the EXO1 synthetic lethality CRISPR screens. ( A ) Diagram showing the overlap of identical genes within the top synthetic lethality hits in the two EXO1-knockout cell lines. The number of genes with MAGeCK score lower than 0.005 (left) and 0.015 (right) are shown. ( B ) The number of common genes within the top synthetic lethality hits in the two EXO1-knockout cell lines compared to the random probability of identical hits. The top hits with MAGeCK score lower than 0.005 (left) and 0.015 (right) were included in the analysis. ( C ) Functional annotation clustering of the common genes within the top synthetic lethality hits with MAGeCK score lower than 0.015 in the two EXO1-knockout cell lines, using Gene Ontology and Uniprot terms. ( D ) Diagram showing the overlap of identical genes within the top synthetic lethality hits in EXO1 KO#1 cells (compared to WT) and the control comparison to EXO1 KO#3 . The number of genes with MAGeCK score lower than 0.005 (left) and 0.015 (right) are shown. ( E ) The number of common genes within the top synthetic lethality hits in EXO1 KO#1 cells (compared to WT) and the control comparison to EXO1 KO#3 . The top hits with MAGeCK score lower than 0.005 (left) and 0.015 (right) were included in the analysis.

    Journal: Nucleic Acids Research

    Article Title: Genome-wide CRISPR screens identify the EXO1-CAF-1 pathway suppressing R-loop-associated DNA damage

    doi: 10.1093/nar/gkag226

    Figure Lengend Snippet: Analyses of the EXO1 synthetic lethality CRISPR screens. ( A ) Diagram showing the overlap of identical genes within the top synthetic lethality hits in the two EXO1-knockout cell lines. The number of genes with MAGeCK score lower than 0.005 (left) and 0.015 (right) are shown. ( B ) The number of common genes within the top synthetic lethality hits in the two EXO1-knockout cell lines compared to the random probability of identical hits. The top hits with MAGeCK score lower than 0.005 (left) and 0.015 (right) were included in the analysis. ( C ) Functional annotation clustering of the common genes within the top synthetic lethality hits with MAGeCK score lower than 0.015 in the two EXO1-knockout cell lines, using Gene Ontology and Uniprot terms. ( D ) Diagram showing the overlap of identical genes within the top synthetic lethality hits in EXO1 KO#1 cells (compared to WT) and the control comparison to EXO1 KO#3 . The number of genes with MAGeCK score lower than 0.005 (left) and 0.015 (right) are shown. ( E ) The number of common genes within the top synthetic lethality hits in EXO1 KO#1 cells (compared to WT) and the control comparison to EXO1 KO#3 . The top hits with MAGeCK score lower than 0.005 (left) and 0.015 (right) were included in the analysis.

    Article Snippet: For CRISPR knockout screens, the Brunello Human CRISPR knockout pooled lentiviral library (Addgene 73179) was used [ ].

    Techniques: CRISPR, Knock-Out, Functional Assay, Control, Comparison

    Co-depletion of EXO1 and CHAF1A reduces cellular viability. ( A, C, E ). Volcano plots showing the results of genome-wide CRISPR knockout screens to identify EXO1 synthetic lethality interactions. Genes targeted by the library are presented based on their impact on the viability of EXO1 KO#1 compared to WT cells ( A ), EXO1 KO#3 compared to WT cells ( C ), and as control, EXO1 KO#1 compared to EXO1 KO#3 cells ( E ). Genes are plotted by the −log 10 of their respective negative and positive P -values and associated log 2 Fold Change values. The hit chosen for validation, namely CHAF1A, is indicated. ( B, D, F ) Scatterplots showing the results of genome-wide CRISPR knockout screens to identify EXO1 synthetic lethality interactions. Genes targeted by the library are plotted based on their impact on the viability of EXO1 KO#1 compared to WT cells ( B ), EXO1 KO#3 compared to WT cells ( D ), and as control, EXO1 KO#1 compared to EXO1 KO#3 cells ( F ). The hit chosen for validation, namely CHAF1A, is indicated. ( G ) Table showing the ranks in the synthetic lethality screens, and the biological roles of CHAF1A. ( H, I ) Clonogenic survival assays showing that siRNA ( H ) and sgRNA ( I ) depletion of CHAF1A reduces the viability of EXO1-knockout cells compared to WT HeLa cells. Clonogenic survival is presented normalized to WT control cells. The average of three independent experiments, with standard deviations indicated as error bars, is shown. Asterisks indicate statistical significance ( t -test unpaired).

    Journal: Nucleic Acids Research

    Article Title: Genome-wide CRISPR screens identify the EXO1-CAF-1 pathway suppressing R-loop-associated DNA damage

    doi: 10.1093/nar/gkag226

    Figure Lengend Snippet: Co-depletion of EXO1 and CHAF1A reduces cellular viability. ( A, C, E ). Volcano plots showing the results of genome-wide CRISPR knockout screens to identify EXO1 synthetic lethality interactions. Genes targeted by the library are presented based on their impact on the viability of EXO1 KO#1 compared to WT cells ( A ), EXO1 KO#3 compared to WT cells ( C ), and as control, EXO1 KO#1 compared to EXO1 KO#3 cells ( E ). Genes are plotted by the −log 10 of their respective negative and positive P -values and associated log 2 Fold Change values. The hit chosen for validation, namely CHAF1A, is indicated. ( B, D, F ) Scatterplots showing the results of genome-wide CRISPR knockout screens to identify EXO1 synthetic lethality interactions. Genes targeted by the library are plotted based on their impact on the viability of EXO1 KO#1 compared to WT cells ( B ), EXO1 KO#3 compared to WT cells ( D ), and as control, EXO1 KO#1 compared to EXO1 KO#3 cells ( F ). The hit chosen for validation, namely CHAF1A, is indicated. ( G ) Table showing the ranks in the synthetic lethality screens, and the biological roles of CHAF1A. ( H, I ) Clonogenic survival assays showing that siRNA ( H ) and sgRNA ( I ) depletion of CHAF1A reduces the viability of EXO1-knockout cells compared to WT HeLa cells. Clonogenic survival is presented normalized to WT control cells. The average of three independent experiments, with standard deviations indicated as error bars, is shown. Asterisks indicate statistical significance ( t -test unpaired).

    Article Snippet: For CRISPR knockout screens, the Brunello Human CRISPR knockout pooled lentiviral library (Addgene 73179) was used [ ].

    Techniques: Genome Wide, CRISPR, Knock-Out, Control, Biomarker Discovery

    ( a ) Schematic view of ex vivo CRISPR/Cas9 screening in mouse primary CD8 + T-cells. ( b ) Volcano plot showing results of ex vivo CRISPR/Cas9 genome-wide screenings. The screenings were repeated independently once. The p-values were calculated using the α-robust rank aggregation (α-RRA) algorithm in MAGeCK. ( c ) Verification of candidate genes by individual single gRNAs. The relative expression levels of surface PD-1 protein and PD-1 mRNA were measured by FACS as mean fluorescent intensity (MFI) and RT-qPCR, respectively. The verification assays were biologically replicated twice. ( d ) GSEA of significantly enriched KEGG pathways in genome-wide screening. The enrichment score (ES) and statistical significance were calculated using the clusterProfiler (version 3.12.0) R package.

    Journal: eLife

    Article Title: Ex vivo and in vivo CRISPR/Cas9 screenings identify the roles of protein N-glycosylation in regulating T-cell activation and functions

    doi: 10.7554/eLife.108724

    Figure Lengend Snippet: ( a ) Schematic view of ex vivo CRISPR/Cas9 screening in mouse primary CD8 + T-cells. ( b ) Volcano plot showing results of ex vivo CRISPR/Cas9 genome-wide screenings. The screenings were repeated independently once. The p-values were calculated using the α-robust rank aggregation (α-RRA) algorithm in MAGeCK. ( c ) Verification of candidate genes by individual single gRNAs. The relative expression levels of surface PD-1 protein and PD-1 mRNA were measured by FACS as mean fluorescent intensity (MFI) and RT-qPCR, respectively. The verification assays were biologically replicated twice. ( d ) GSEA of significantly enriched KEGG pathways in genome-wide screening. The enrichment score (ES) and statistical significance were calculated using the clusterProfiler (version 3.12.0) R package.

    Article Snippet: A whole-genome CRISPR knockout gRNA library (1000000096) was purchased from Addgene.

    Techniques: Ex Vivo, CRISPR, Genome Wide, Expressing, Quantitative RT-PCR

    ( a ) Schematic view of in vivo CRISPR/Cas9 screening in mouse primary CD8 + T-cells. ( b ) Volcano plot showing results of ex vivo CRISPR/Cas9 screening. The screenings were repeated independently once. The p-values were calculated using the α-RRA algorithm in MAGeCK. ( c ) Volcano plot showing results of in vivo CRISPR/Cas9 screenings. The screenings were repeated independently once. The p-values were calculated using the α-RRA algorithm in MAGeCK.

    Journal: eLife

    Article Title: Ex vivo and in vivo CRISPR/Cas9 screenings identify the roles of protein N-glycosylation in regulating T-cell activation and functions

    doi: 10.7554/eLife.108724

    Figure Lengend Snippet: ( a ) Schematic view of in vivo CRISPR/Cas9 screening in mouse primary CD8 + T-cells. ( b ) Volcano plot showing results of ex vivo CRISPR/Cas9 screening. The screenings were repeated independently once. The p-values were calculated using the α-RRA algorithm in MAGeCK. ( c ) Volcano plot showing results of in vivo CRISPR/Cas9 screenings. The screenings were repeated independently once. The p-values were calculated using the α-RRA algorithm in MAGeCK.

    Article Snippet: A whole-genome CRISPR knockout gRNA library (1000000096) was purchased from Addgene.

    Techniques: In Vivo, CRISPR, Ex Vivo

    ( a ) CRISPR/Cas9 knockout of B4galt1 (sgB4galt1) (sg2) in CD8 + T-cells increases expression of PD-1 before and after co-culture with B16F10-OVA cells. The MFIs of PD-1 were measured by FACS (n=6). The relative mRNA levels of PD-1 were measured by quantitative RT-qPCR (n=6). The p-values were calculated using a two-tailed Student’s t -test. ( b ) The effect of B4galt1 knockout on PD-1 surface expression could be rescued by overexpression of either long- or short-isoform B4galt1 (n=3). The p-values were calculated using a two-tailed Student’s t -test. ( c ) CRISPR/Cas9 knockout of B4galt1 in CD8 + T-cells increases expression of TNFα and IFNγ after co-culture with B16F10-OVA cells. The relative mRNA levels were measured by quantitative RT-qPCR (n=3). The secreted TNFα and IFNγ in medium were measured by ELISA (n=6). The p-values were calculated using a two-tailed Student’s t -test. ( d ) CRISPR/Cas9 knockout of B4galt1 in OT-I CD8 + T-cells increases in vitro specific killing activities on B16F10-OVA cells (n=3). The p-values were calculated using a two-tailed Student’s t -test. ( e ) Schematic view of B4GALT1 knockdown in human NY-ESO-1 TCR-T-cells. ( f ) Knockdown of B4GALT1 in human NY-ESO-1 TCR-T-cells by shRNA increases in vitro killing activities on A375 cells (n=5). The p-values were calculated using a two-tailed Student’s t -test. ( g ) Knockdown of B4GALT1 in human NY-ESO-1 TCR-T-cells increases expression of TNFα and IFNγ after co-culture with A375 cells. The secreted TNFα and IFNγ in medium were measured by ELISA (n=3). The p-values were calculated using a two-tailed Student’s t -test. ( h ) Heatmap demonstrating differentially expressed genes (DEGs) between B4galt1 knockout and control mouse OT-I CD8 + T-cells after co-culture. The genes in TCR signaling pathway are labeled on the left side. ( i ) Volcano plot showing upregulated and downregulated genes (p-value <0.01) in B4galt1 knockout mouse OT-I CD8 + T-cells after co-culture. The genes in TCR signaling pathway are labeled with dark blue and dark red. Top genes and some genes in TCR signaling pathway are annotated. The p-value was calculated using the Wald test, and p.adjust was calculated using Benjamini–Hochberg with the R package DESeq2 (version 1.22.2). ( j ) Bar graph showing KEGG pathways significantly changed in B4galt1 knockout mouse OT-I CD8 + T-cells after co-culture. The p-value was calculated using the clusterProfiler (version 3.12.0) R package. All of these functional effects were biologically replicated at least twice. Data are shown as the mean ± SEM. *p<0.05; **p<0.01; ***p<0.001.

    Journal: eLife

    Article Title: Ex vivo and in vivo CRISPR/Cas9 screenings identify the roles of protein N-glycosylation in regulating T-cell activation and functions

    doi: 10.7554/eLife.108724

    Figure Lengend Snippet: ( a ) CRISPR/Cas9 knockout of B4galt1 (sgB4galt1) (sg2) in CD8 + T-cells increases expression of PD-1 before and after co-culture with B16F10-OVA cells. The MFIs of PD-1 were measured by FACS (n=6). The relative mRNA levels of PD-1 were measured by quantitative RT-qPCR (n=6). The p-values were calculated using a two-tailed Student’s t -test. ( b ) The effect of B4galt1 knockout on PD-1 surface expression could be rescued by overexpression of either long- or short-isoform B4galt1 (n=3). The p-values were calculated using a two-tailed Student’s t -test. ( c ) CRISPR/Cas9 knockout of B4galt1 in CD8 + T-cells increases expression of TNFα and IFNγ after co-culture with B16F10-OVA cells. The relative mRNA levels were measured by quantitative RT-qPCR (n=3). The secreted TNFα and IFNγ in medium were measured by ELISA (n=6). The p-values were calculated using a two-tailed Student’s t -test. ( d ) CRISPR/Cas9 knockout of B4galt1 in OT-I CD8 + T-cells increases in vitro specific killing activities on B16F10-OVA cells (n=3). The p-values were calculated using a two-tailed Student’s t -test. ( e ) Schematic view of B4GALT1 knockdown in human NY-ESO-1 TCR-T-cells. ( f ) Knockdown of B4GALT1 in human NY-ESO-1 TCR-T-cells by shRNA increases in vitro killing activities on A375 cells (n=5). The p-values were calculated using a two-tailed Student’s t -test. ( g ) Knockdown of B4GALT1 in human NY-ESO-1 TCR-T-cells increases expression of TNFα and IFNγ after co-culture with A375 cells. The secreted TNFα and IFNγ in medium were measured by ELISA (n=3). The p-values were calculated using a two-tailed Student’s t -test. ( h ) Heatmap demonstrating differentially expressed genes (DEGs) between B4galt1 knockout and control mouse OT-I CD8 + T-cells after co-culture. The genes in TCR signaling pathway are labeled on the left side. ( i ) Volcano plot showing upregulated and downregulated genes (p-value <0.01) in B4galt1 knockout mouse OT-I CD8 + T-cells after co-culture. The genes in TCR signaling pathway are labeled with dark blue and dark red. Top genes and some genes in TCR signaling pathway are annotated. The p-value was calculated using the Wald test, and p.adjust was calculated using Benjamini–Hochberg with the R package DESeq2 (version 1.22.2). ( j ) Bar graph showing KEGG pathways significantly changed in B4galt1 knockout mouse OT-I CD8 + T-cells after co-culture. The p-value was calculated using the clusterProfiler (version 3.12.0) R package. All of these functional effects were biologically replicated at least twice. Data are shown as the mean ± SEM. *p<0.05; **p<0.01; ***p<0.001.

    Article Snippet: A whole-genome CRISPR knockout gRNA library (1000000096) was purchased from Addgene.

    Techniques: CRISPR, Knock-Out, Expressing, Co-Culture Assay, Quantitative RT-PCR, Two Tailed Test, Over Expression, Enzyme-linked Immunosorbent Assay, In Vitro, Knockdown, shRNA, Control, Labeling, Functional Assay

    CRISPR/Cas9 knockout of B4GALT1 in hCD19-CAR-T-cells does not affect in vitro killing of Nalm6 target cells (n=3). The killing assays were biologically replicated three times. Data are shown as the mean ± SEM. NS, not significant.

    Journal: eLife

    Article Title: Ex vivo and in vivo CRISPR/Cas9 screenings identify the roles of protein N-glycosylation in regulating T-cell activation and functions

    doi: 10.7554/eLife.108724

    Figure Lengend Snippet: CRISPR/Cas9 knockout of B4GALT1 in hCD19-CAR-T-cells does not affect in vitro killing of Nalm6 target cells (n=3). The killing assays were biologically replicated three times. Data are shown as the mean ± SEM. NS, not significant.

    Article Snippet: A whole-genome CRISPR knockout gRNA library (1000000096) was purchased from Addgene.

    Techniques: CRISPR, Knock-Out, In Vitro

    ( a ) Schematic view of B4galt1 functional test in tumor microenvironment. ( b ) CRISPR/Cas9 knockout of B4galt1 in OT-I T-cells enhances growth control of B16F10-OVA tumors in vivo. The p-value was calculated using two-way ANOVA. ( c ) Compared with control OT-I T-cells, the tumors were significantly smaller when B4galt1 knockout OT-I T-cells were transplanted (n=5 for control, n=7 for sgB4galt1). The p-value was calculated using a two-tailed Student’s t -test. ( d ) CRISPR/Cas9 knockout of B4galt1 increases numbers of OT-I T-cells in B16F10-OVA tumors (n=5 for control, n=7 for sgB4galt1). The p-value was calculated using a two-tailed Student’s t -test. The in vivo functional effects were biologically replicated at least twice. Data are shown as the mean ± SEM. *p<0.05; **p<0.01.

    Journal: eLife

    Article Title: Ex vivo and in vivo CRISPR/Cas9 screenings identify the roles of protein N-glycosylation in regulating T-cell activation and functions

    doi: 10.7554/eLife.108724

    Figure Lengend Snippet: ( a ) Schematic view of B4galt1 functional test in tumor microenvironment. ( b ) CRISPR/Cas9 knockout of B4galt1 in OT-I T-cells enhances growth control of B16F10-OVA tumors in vivo. The p-value was calculated using two-way ANOVA. ( c ) Compared with control OT-I T-cells, the tumors were significantly smaller when B4galt1 knockout OT-I T-cells were transplanted (n=5 for control, n=7 for sgB4galt1). The p-value was calculated using a two-tailed Student’s t -test. ( d ) CRISPR/Cas9 knockout of B4galt1 increases numbers of OT-I T-cells in B16F10-OVA tumors (n=5 for control, n=7 for sgB4galt1). The p-value was calculated using a two-tailed Student’s t -test. The in vivo functional effects were biologically replicated at least twice. Data are shown as the mean ± SEM. *p<0.05; **p<0.01.

    Article Snippet: A whole-genome CRISPR knockout gRNA library (1000000096) was purchased from Addgene.

    Techniques: Functional Assay, CRISPR, Knock-Out, Control, In Vivo, Two Tailed Test