lenticrisprv2 Addgene Inc Search Results


  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 99
    Addgene inc lenticrisprv2
    GFP-Tsg101 knock-in using CRISPR-Cas9 and HDR. A , knock-in strategy. Cells were transduced with <t>LentiCRISPRv2</t> to express Cas9 with sgRNA targeting the start of the Tsg101 gene and transfected with a donor template plasmid containing a puromycin resistance selection marker and a GFP tag flanked by HRs for the target site. Cas9 generates a DSB in the genomic DNA at the target site, and the donor template can be integrated into the DSB by HDR, resulting in GFP knock-in. Otherwise, the DSB is repaired by the more efficient process of NHEJ, leaving insertions or deletions that knock out Tsg101. Tsg101 knockout was lethal, which helped to select against cells that did not achieve the knock-in. B , Western blotting for Tsg101 in cell lysates of GFP-Tsg101 KI clonal lines and WT parental lines. The KI cell lines express GFP-Tsg101 at lower than WT levels and do not express untagged Tsg101. α-Tubulin is shown as a loading control.
    Lenticrisprv2, supplied by Addgene inc, used in various techniques. Bioz Stars score: 99/100, based on 1220 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/lenticrisprv2/product/Addgene inc
    Average 99 stars, based on 1220 article reviews
    Price from $9.99 to $1999.99
    lenticrisprv2 - by Bioz Stars, 2020-02
    99/100 stars
      Buy from Supplier

    90
    Addgene inc bsmbi digested lenticrisprv2
    GFP-Tsg101 knock-in using CRISPR-Cas9 and HDR. A , knock-in strategy. Cells were transduced with <t>LentiCRISPRv2</t> to express Cas9 with sgRNA targeting the start of the Tsg101 gene and transfected with a donor template plasmid containing a puromycin resistance selection marker and a GFP tag flanked by HRs for the target site. Cas9 generates a DSB in the genomic DNA at the target site, and the donor template can be integrated into the DSB by HDR, resulting in GFP knock-in. Otherwise, the DSB is repaired by the more efficient process of NHEJ, leaving insertions or deletions that knock out Tsg101. Tsg101 knockout was lethal, which helped to select against cells that did not achieve the knock-in. B , Western blotting for Tsg101 in cell lysates of GFP-Tsg101 KI clonal lines and WT parental lines. The KI cell lines express GFP-Tsg101 at lower than WT levels and do not express untagged Tsg101. α-Tubulin is shown as a loading control.
    Bsmbi Digested Lenticrisprv2, supplied by Addgene inc, used in various techniques. Bioz Stars score: 90/100, based on 25 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/bsmbi digested lenticrisprv2/product/Addgene inc
    Average 90 stars, based on 25 article reviews
    Price from $9.99 to $1999.99
    bsmbi digested lenticrisprv2 - by Bioz Stars, 2020-02
    90/100 stars
      Buy from Supplier

    82
    Addgene inc lenticrisprv2 a9 cells lenticrisprv2 plasmid
    MVM associates with artificially-engineered sites of DNA damage. ( A ) Murine A9 fibroblasts were infected with MVM at an MOI of 10 for 18 hr. Cells were sensitized with Hoechst 33342 solution prior to irradiation in selected regions of interest with a 405 nanometer laser (top 3 panels), and were compared with un-irradiated cells (bottom panel). Cells were fixed and analyzed for the localization of DNA damaged sites (detected by green γ-H2AX staining), with that of MVM NS1 protein ( red ). The irrelevant transcription factor NR5A2 ( red ) was used as a negative control as this marker that does not colocalize with APAR bodies (see Figure 1 ). The nuclear periphery is demarcated with dotted white lines and DAPI staining. ( B ) Schematic of γ-H2AX binding to the cellular genome on chromosome 9 (top panel) compared with MVM-interaction sites (bottom panel) in parasynchronized murine <t>A9</t> cells infected with MVM (MOI 5) for 16 hr. The site where guide RNAs were designed for DDR induction is shown as a red rectangle and labelled ‘CRISPR/Cas9’. Focused V3C-qPCR assays were performed in A9 cells constitutively expressing <t>LentiCRISPRv2</t> which were transiently transfected with guide RNAs (nontargeting, labelled as ‘CTRL’ or specific to the 9qE1 site, labelled as ‘TGT’), and infected with MVM at an MOI for 5 for 16 hr. The spatial association of MVM with cellular sites were determined with Taqman probes on ( C ), the MVM genome, and reciprocal interaction with the Taqman probe on ( D ), the 9qE1 break site. ( E ) MVM association with Chr19 VAD at 19qA was tested by focused Taqman qPCR. NS1 occupancy at ( F ), the 9qE1 break site and ( G ), the 19qA VAD was assayed by ChIP-qPCR in cells transfected with non-targeting (CTRL) and specific (TGT) guide RNAs. Background levels were determined using IgG pulldowns. qPCR data are presented as mean ± SEM of three independent experiments.
    Lenticrisprv2 A9 Cells Lenticrisprv2 Plasmid, supplied by Addgene inc, used in various techniques. Bioz Stars score: 82/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/lenticrisprv2 a9 cells lenticrisprv2 plasmid/product/Addgene inc
    Average 82 stars, based on 6 article reviews
    Price from $9.99 to $1999.99
    lenticrisprv2 a9 cells lenticrisprv2 plasmid - by Bioz Stars, 2020-02
    82/100 stars
      Buy from Supplier

    99
    Addgene inc lenticrisprv2 plasmid
    Optimization of 2CT-CRISPR assay system for genome-scale screening a, Representative FACS plot of B2M expression in Mel624 cells on day 5 after transduction with <t>lentiCRISPRv2</t> lentivirus containing a pool of three sgRNAs targeting B2M . b–c, Cas9 disruption of MHC Class I antigen presentation/processing pathway genes reduces efficacy of T cell-mediated cytolysis. Timeline shows 12 hours of co-culture of ESO T cells with individual gene edited Mel624 cells at E:T ratio of 0.5. Live cell survival (%) was calculated from control cells unexposed to T cell selection. Each dot in the plot represents independent gene-specific CRISPR lentivirus infection replicate ( n = 3). Improvement in CRISPR edited cell yields at 60 h timepoint compared to 36 h after 2CT assay as shown in ( c) . All values are mean ± s.e.m. Data is representative of two independent experiments.
    Lenticrisprv2 Plasmid, supplied by Addgene inc, used in various techniques. Bioz Stars score: 99/100, based on 586 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/lenticrisprv2 plasmid/product/Addgene inc
    Average 99 stars, based on 586 article reviews
    Price from $9.99 to $1999.99
    lenticrisprv2 plasmid - by Bioz Stars, 2020-02
    99/100 stars
      Buy from Supplier

    84
    Addgene inc bacterial dna carrying empty lenticrisprv2 vectors
    Optimization of 2CT-CRISPR assay system for genome-scale screening a, Representative FACS plot of B2M expression in Mel624 cells on day 5 after transduction with <t>lentiCRISPRv2</t> lentivirus containing a pool of three sgRNAs targeting B2M . b–c, Cas9 disruption of MHC Class I antigen presentation/processing pathway genes reduces efficacy of T cell-mediated cytolysis. Timeline shows 12 hours of co-culture of ESO T cells with individual gene edited Mel624 cells at E:T ratio of 0.5. Live cell survival (%) was calculated from control cells unexposed to T cell selection. Each dot in the plot represents independent gene-specific CRISPR lentivirus infection replicate ( n = 3). Improvement in CRISPR edited cell yields at 60 h timepoint compared to 36 h after 2CT assay as shown in ( c) . All values are mean ± s.e.m. Data is representative of two independent experiments.
    Bacterial Dna Carrying Empty Lenticrisprv2 Vectors, supplied by Addgene inc, used in various techniques. Bioz Stars score: 84/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/bacterial dna carrying empty lenticrisprv2 vectors/product/Addgene inc
    Average 84 stars, based on 5 article reviews
    Price from $9.99 to $1999.99
    bacterial dna carrying empty lenticrisprv2 vectors - by Bioz Stars, 2020-02
    84/100 stars
      Buy from Supplier

    85
    Addgene inc bsmbi digested lenticrisprv2 plasmid
    Optimization of 2CT-CRISPR assay system for genome-scale screening a, Representative FACS plot of B2M expression in Mel624 cells on day 5 after transduction with <t>lentiCRISPRv2</t> lentivirus containing a pool of three sgRNAs targeting B2M . b–c, Cas9 disruption of MHC Class I antigen presentation/processing pathway genes reduces efficacy of T cell-mediated cytolysis. Timeline shows 12 hours of co-culture of ESO T cells with individual gene edited Mel624 cells at E:T ratio of 0.5. Live cell survival (%) was calculated from control cells unexposed to T cell selection. Each dot in the plot represents independent gene-specific CRISPR lentivirus infection replicate ( n = 3). Improvement in CRISPR edited cell yields at 60 h timepoint compared to 36 h after 2CT assay as shown in ( c) . All values are mean ± s.e.m. Data is representative of two independent experiments.
    Bsmbi Digested Lenticrisprv2 Plasmid, supplied by Addgene inc, used in various techniques. Bioz Stars score: 85/100, based on 17 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/bsmbi digested lenticrisprv2 plasmid/product/Addgene inc
    Average 85 stars, based on 17 article reviews
    Price from $9.99 to $1999.99
    bsmbi digested lenticrisprv2 plasmid - by Bioz Stars, 2020-02
    85/100 stars
      Buy from Supplier

    79
    Addgene inc all in one expression plasmid lenticrisprv2
    Optimization of 2CT-CRISPR assay system for genome-scale screening a, Representative FACS plot of B2M expression in Mel624 cells on day 5 after transduction with <t>lentiCRISPRv2</t> lentivirus containing a pool of three sgRNAs targeting B2M . b–c, Cas9 disruption of MHC Class I antigen presentation/processing pathway genes reduces efficacy of T cell-mediated cytolysis. Timeline shows 12 hours of co-culture of ESO T cells with individual gene edited Mel624 cells at E:T ratio of 0.5. Live cell survival (%) was calculated from control cells unexposed to T cell selection. Each dot in the plot represents independent gene-specific CRISPR lentivirus infection replicate ( n = 3). Improvement in CRISPR edited cell yields at 60 h timepoint compared to 36 h after 2CT assay as shown in ( c) . All values are mean ± s.e.m. Data is representative of two independent experiments.
    All In One Expression Plasmid Lenticrisprv2, supplied by Addgene inc, used in various techniques. Bioz Stars score: 79/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/all in one expression plasmid lenticrisprv2/product/Addgene inc
    Average 79 stars, based on 5 article reviews
    Price from $9.99 to $1999.99
    all in one expression plasmid lenticrisprv2 - by Bioz Stars, 2020-02
    79/100 stars
      Buy from Supplier

    80
    Addgene inc lenticrisprv2 lcv2 vector
    Optimization of 2CT-CRISPR assay system for genome-scale screening a, Representative FACS plot of B2M expression in Mel624 cells on day 5 after transduction with <t>lentiCRISPRv2</t> lentivirus containing a pool of three sgRNAs targeting B2M . b–c, Cas9 disruption of MHC Class I antigen presentation/processing pathway genes reduces efficacy of T cell-mediated cytolysis. Timeline shows 12 hours of co-culture of ESO T cells with individual gene edited Mel624 cells at E:T ratio of 0.5. Live cell survival (%) was calculated from control cells unexposed to T cell selection. Each dot in the plot represents independent gene-specific CRISPR lentivirus infection replicate ( n = 3). Improvement in CRISPR edited cell yields at 60 h timepoint compared to 36 h after 2CT assay as shown in ( c) . All values are mean ± s.e.m. Data is representative of two independent experiments.
    Lenticrisprv2 Lcv2 Vector, supplied by Addgene inc, used in various techniques. Bioz Stars score: 80/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/lenticrisprv2 lcv2 vector/product/Addgene inc
    Average 80 stars, based on 4 article reviews
    Price from $9.99 to $1999.99
    lenticrisprv2 lcv2 vector - by Bioz Stars, 2020-02
    80/100 stars
      Buy from Supplier

    79
    Addgene inc lenticrisprv2 puro system
    Optimization of 2CT-CRISPR assay system for genome-scale screening a, Representative FACS plot of B2M expression in Mel624 cells on day 5 after transduction with <t>lentiCRISPRv2</t> lentivirus containing a pool of three sgRNAs targeting B2M . b–c, Cas9 disruption of MHC Class I antigen presentation/processing pathway genes reduces efficacy of T cell-mediated cytolysis. Timeline shows 12 hours of co-culture of ESO T cells with individual gene edited Mel624 cells at E:T ratio of 0.5. Live cell survival (%) was calculated from control cells unexposed to T cell selection. Each dot in the plot represents independent gene-specific CRISPR lentivirus infection replicate ( n = 3). Improvement in CRISPR edited cell yields at 60 h timepoint compared to 36 h after 2CT assay as shown in ( c) . All values are mean ± s.e.m. Data is representative of two independent experiments.
    Lenticrisprv2 Puro System, supplied by Addgene inc, used in various techniques. Bioz Stars score: 79/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/lenticrisprv2 puro system/product/Addgene inc
    Average 79 stars, based on 3 article reviews
    Price from $9.99 to $1999.99
    lenticrisprv2 puro system - by Bioz Stars, 2020-02
    79/100 stars
      Buy from Supplier

    89
    Addgene inc lenticrisprv2 grna construct pspax2
    Optimization of 2CT-CRISPR assay system for genome-scale screening a, Representative FACS plot of B2M expression in Mel624 cells on day 5 after transduction with <t>lentiCRISPRv2</t> lentivirus containing a pool of three sgRNAs targeting B2M . b–c, Cas9 disruption of MHC Class I antigen presentation/processing pathway genes reduces efficacy of T cell-mediated cytolysis. Timeline shows 12 hours of co-culture of ESO T cells with individual gene edited Mel624 cells at E:T ratio of 0.5. Live cell survival (%) was calculated from control cells unexposed to T cell selection. Each dot in the plot represents independent gene-specific CRISPR lentivirus infection replicate ( n = 3). Improvement in CRISPR edited cell yields at 60 h timepoint compared to 36 h after 2CT assay as shown in ( c) . All values are mean ± s.e.m. Data is representative of two independent experiments.
    Lenticrisprv2 Grna Construct Pspax2, supplied by Addgene inc, used in various techniques. Bioz Stars score: 89/100, based on 16 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/lenticrisprv2 grna construct pspax2/product/Addgene inc
    Average 89 stars, based on 16 article reviews
    Price from $9.99 to $1999.99
    lenticrisprv2 grna construct pspax2 - by Bioz Stars, 2020-02
    89/100 stars
      Buy from Supplier

    91
    Addgene inc lenticrisprv2 sgrna
    Optimization of 2CT-CRISPR assay system for genome-scale screening a, Representative FACS plot of B2M expression in Mel624 cells on day 5 after transduction with <t>lentiCRISPRv2</t> lentivirus containing a pool of three sgRNAs targeting B2M . b–c, Cas9 disruption of MHC Class I antigen presentation/processing pathway genes reduces efficacy of T cell-mediated cytolysis. Timeline shows 12 hours of co-culture of ESO T cells with individual gene edited Mel624 cells at E:T ratio of 0.5. Live cell survival (%) was calculated from control cells unexposed to T cell selection. Each dot in the plot represents independent gene-specific CRISPR lentivirus infection replicate ( n = 3). Improvement in CRISPR edited cell yields at 60 h timepoint compared to 36 h after 2CT assay as shown in ( c) . All values are mean ± s.e.m. Data is representative of two independent experiments.
    Lenticrisprv2 Sgrna, supplied by Addgene inc, used in various techniques. Bioz Stars score: 91/100, based on 31 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/lenticrisprv2 sgrna/product/Addgene inc
    Average 91 stars, based on 31 article reviews
    Price from $9.99 to $1999.99
    lenticrisprv2 sgrna - by Bioz Stars, 2020-02
    91/100 stars
      Buy from Supplier

    78
    Addgene inc plasmid lenticrisprv2 puro
    Optimization of 2CT-CRISPR assay system for genome-scale screening a, Representative FACS plot of B2M expression in Mel624 cells on day 5 after transduction with <t>lentiCRISPRv2</t> lentivirus containing a pool of three sgRNAs targeting B2M . b–c, Cas9 disruption of MHC Class I antigen presentation/processing pathway genes reduces efficacy of T cell-mediated cytolysis. Timeline shows 12 hours of co-culture of ESO T cells with individual gene edited Mel624 cells at E:T ratio of 0.5. Live cell survival (%) was calculated from control cells unexposed to T cell selection. Each dot in the plot represents independent gene-specific CRISPR lentivirus infection replicate ( n = 3). Improvement in CRISPR edited cell yields at 60 h timepoint compared to 36 h after 2CT assay as shown in ( c) . All values are mean ± s.e.m. Data is representative of two independent experiments.
    Plasmid Lenticrisprv2 Puro, supplied by Addgene inc, used in various techniques. Bioz Stars score: 78/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/plasmid lenticrisprv2 puro/product/Addgene inc
    Average 78 stars, based on 5 article reviews
    Price from $9.99 to $1999.99
    plasmid lenticrisprv2 puro - by Bioz Stars, 2020-02
    78/100 stars
      Buy from Supplier

    90
    Addgene inc lentivirus expression vector lenticrisprv2
    Optimization of 2CT-CRISPR assay system for genome-scale screening a, Representative FACS plot of B2M expression in Mel624 cells on day 5 after transduction with <t>lentiCRISPRv2</t> lentivirus containing a pool of three sgRNAs targeting B2M . b–c, Cas9 disruption of MHC Class I antigen presentation/processing pathway genes reduces efficacy of T cell-mediated cytolysis. Timeline shows 12 hours of co-culture of ESO T cells with individual gene edited Mel624 cells at E:T ratio of 0.5. Live cell survival (%) was calculated from control cells unexposed to T cell selection. Each dot in the plot represents independent gene-specific CRISPR lentivirus infection replicate ( n = 3). Improvement in CRISPR edited cell yields at 60 h timepoint compared to 36 h after 2CT assay as shown in ( c) . All values are mean ± s.e.m. Data is representative of two independent experiments.
    Lentivirus Expression Vector Lenticrisprv2, supplied by Addgene inc, used in various techniques. Bioz Stars score: 90/100, based on 20 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/lentivirus expression vector lenticrisprv2/product/Addgene inc
    Average 90 stars, based on 20 article reviews
    Price from $9.99 to $1999.99
    lentivirus expression vector lenticrisprv2 - by Bioz Stars, 2020-02
    90/100 stars
      Buy from Supplier

    99
    Addgene inc lentiviral vector lenticrisprv2
    Optimization of 2CT-CRISPR assay system for genome-scale screening a, Representative FACS plot of B2M expression in Mel624 cells on day 5 after transduction with <t>lentiCRISPRv2</t> lentivirus containing a pool of three sgRNAs targeting B2M . b–c, Cas9 disruption of MHC Class I antigen presentation/processing pathway genes reduces efficacy of T cell-mediated cytolysis. Timeline shows 12 hours of co-culture of ESO T cells with individual gene edited Mel624 cells at E:T ratio of 0.5. Live cell survival (%) was calculated from control cells unexposed to T cell selection. Each dot in the plot represents independent gene-specific CRISPR lentivirus infection replicate ( n = 3). Improvement in CRISPR edited cell yields at 60 h timepoint compared to 36 h after 2CT assay as shown in ( c) . All values are mean ± s.e.m. Data is representative of two independent experiments.
    Lentiviral Vector Lenticrisprv2, supplied by Addgene inc, used in various techniques. Bioz Stars score: 99/100, based on 67 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/lentiviral vector lenticrisprv2/product/Addgene inc
    Average 99 stars, based on 67 article reviews
    Price from $9.99 to $1999.99
    lentiviral vector lenticrisprv2 - by Bioz Stars, 2020-02
    99/100 stars
      Buy from Supplier

    85
    Addgene inc cas9 expressing lenticrisprv2 vector
    Optimization of 2CT-CRISPR assay system for genome-scale screening a, Representative FACS plot of B2M expression in Mel624 cells on day 5 after transduction with <t>lentiCRISPRv2</t> lentivirus containing a pool of three sgRNAs targeting B2M . b–c, Cas9 disruption of MHC Class I antigen presentation/processing pathway genes reduces efficacy of T cell-mediated cytolysis. Timeline shows 12 hours of co-culture of ESO T cells with individual gene edited Mel624 cells at E:T ratio of 0.5. Live cell survival (%) was calculated from control cells unexposed to T cell selection. Each dot in the plot represents independent gene-specific CRISPR lentivirus infection replicate ( n = 3). Improvement in CRISPR edited cell yields at 60 h timepoint compared to 36 h after 2CT assay as shown in ( c) . All values are mean ± s.e.m. Data is representative of two independent experiments.
    Cas9 Expressing Lenticrisprv2 Vector, supplied by Addgene inc, used in various techniques. Bioz Stars score: 85/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/cas9 expressing lenticrisprv2 vector/product/Addgene inc
    Average 85 stars, based on 8 article reviews
    Price from $9.99 to $1999.99
    cas9 expressing lenticrisprv2 vector - by Bioz Stars, 2020-02
    85/100 stars
      Buy from Supplier

    83
    Addgene inc lentivirus vector lenticrisprv2
    Optimization of 2CT-CRISPR assay system for genome-scale screening a, Representative FACS plot of B2M expression in Mel624 cells on day 5 after transduction with <t>lentiCRISPRv2</t> lentivirus containing a pool of three sgRNAs targeting B2M . b–c, Cas9 disruption of MHC Class I antigen presentation/processing pathway genes reduces efficacy of T cell-mediated cytolysis. Timeline shows 12 hours of co-culture of ESO T cells with individual gene edited Mel624 cells at E:T ratio of 0.5. Live cell survival (%) was calculated from control cells unexposed to T cell selection. Each dot in the plot represents independent gene-specific CRISPR lentivirus infection replicate ( n = 3). Improvement in CRISPR edited cell yields at 60 h timepoint compared to 36 h after 2CT assay as shown in ( c) . All values are mean ± s.e.m. Data is representative of two independent experiments.
    Lentivirus Vector Lenticrisprv2, supplied by Addgene inc, used in various techniques. Bioz Stars score: 83/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/lentivirus vector lenticrisprv2/product/Addgene inc
    Average 83 stars, based on 6 article reviews
    Price from $9.99 to $1999.99
    lentivirus vector lenticrisprv2 - by Bioz Stars, 2020-02
    83/100 stars
      Buy from Supplier

    79
    Addgene inc lenticrisprv2 lentiviral vector system
    Optimization of 2CT-CRISPR assay system for genome-scale screening a, Representative FACS plot of B2M expression in Mel624 cells on day 5 after transduction with <t>lentiCRISPRv2</t> lentivirus containing a pool of three sgRNAs targeting B2M . b–c, Cas9 disruption of MHC Class I antigen presentation/processing pathway genes reduces efficacy of T cell-mediated cytolysis. Timeline shows 12 hours of co-culture of ESO T cells with individual gene edited Mel624 cells at E:T ratio of 0.5. Live cell survival (%) was calculated from control cells unexposed to T cell selection. Each dot in the plot represents independent gene-specific CRISPR lentivirus infection replicate ( n = 3). Improvement in CRISPR edited cell yields at 60 h timepoint compared to 36 h after 2CT assay as shown in ( c) . All values are mean ± s.e.m. Data is representative of two independent experiments.
    Lenticrisprv2 Lentiviral Vector System, supplied by Addgene inc, used in various techniques. Bioz Stars score: 79/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/lenticrisprv2 lentiviral vector system/product/Addgene inc
    Average 79 stars, based on 5 article reviews
    Price from $9.99 to $1999.99
    lenticrisprv2 lentiviral vector system - by Bioz Stars, 2020-02
    79/100 stars
      Buy from Supplier

    85
    Addgene inc alkaline phosphatase digested lenticrisprv2 blast
    Optimization of 2CT-CRISPR assay system for genome-scale screening a, Representative FACS plot of B2M expression in Mel624 cells on day 5 after transduction with <t>lentiCRISPRv2</t> lentivirus containing a pool of three sgRNAs targeting B2M . b–c, Cas9 disruption of MHC Class I antigen presentation/processing pathway genes reduces efficacy of T cell-mediated cytolysis. Timeline shows 12 hours of co-culture of ESO T cells with individual gene edited Mel624 cells at E:T ratio of 0.5. Live cell survival (%) was calculated from control cells unexposed to T cell selection. Each dot in the plot represents independent gene-specific CRISPR lentivirus infection replicate ( n = 3). Improvement in CRISPR edited cell yields at 60 h timepoint compared to 36 h after 2CT assay as shown in ( c) . All values are mean ± s.e.m. Data is representative of two independent experiments.
    Alkaline Phosphatase Digested Lenticrisprv2 Blast, supplied by Addgene inc, used in various techniques. Bioz Stars score: 85/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/alkaline phosphatase digested lenticrisprv2 blast/product/Addgene inc
    Average 85 stars, based on 8 article reviews
    Price from $9.99 to $1999.99
    alkaline phosphatase digested lenticrisprv2 blast - by Bioz Stars, 2020-02
    85/100 stars
      Buy from Supplier

    79
    Addgene inc human lenticrisprv2 plasmid library
    Optimization of 2CT-CRISPR assay system for genome-scale screening a, Representative FACS plot of B2M expression in Mel624 cells on day 5 after transduction with <t>lentiCRISPRv2</t> lentivirus containing a pool of three sgRNAs targeting B2M . b–c, Cas9 disruption of MHC Class I antigen presentation/processing pathway genes reduces efficacy of T cell-mediated cytolysis. Timeline shows 12 hours of co-culture of ESO T cells with individual gene edited Mel624 cells at E:T ratio of 0.5. Live cell survival (%) was calculated from control cells unexposed to T cell selection. Each dot in the plot represents independent gene-specific CRISPR lentivirus infection replicate ( n = 3). Improvement in CRISPR edited cell yields at 60 h timepoint compared to 36 h after 2CT assay as shown in ( c) . All values are mean ± s.e.m. Data is representative of two independent experiments.
    Human Lenticrisprv2 Plasmid Library, supplied by Addgene inc, used in various techniques. Bioz Stars score: 79/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/human lenticrisprv2 plasmid library/product/Addgene inc
    Average 79 stars, based on 3 article reviews
    Price from $9.99 to $1999.99
    human lenticrisprv2 plasmid library - by Bioz Stars, 2020-02
    79/100 stars
      Buy from Supplier

    81
    Addgene inc bbsi digested lenticrisprv2 control vector
    Optimization of 2CT-CRISPR assay system for genome-scale screening a, Representative FACS plot of B2M expression in Mel624 cells on day 5 after transduction with <t>lentiCRISPRv2</t> lentivirus containing a pool of three sgRNAs targeting B2M . b–c, Cas9 disruption of MHC Class I antigen presentation/processing pathway genes reduces efficacy of T cell-mediated cytolysis. Timeline shows 12 hours of co-culture of ESO T cells with individual gene edited Mel624 cells at E:T ratio of 0.5. Live cell survival (%) was calculated from control cells unexposed to T cell selection. Each dot in the plot represents independent gene-specific CRISPR lentivirus infection replicate ( n = 3). Improvement in CRISPR edited cell yields at 60 h timepoint compared to 36 h after 2CT assay as shown in ( c) . All values are mean ± s.e.m. Data is representative of two independent experiments.
    Bbsi Digested Lenticrisprv2 Control Vector, supplied by Addgene inc, used in various techniques. Bioz Stars score: 81/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/bbsi digested lenticrisprv2 control vector/product/Addgene inc
    Average 81 stars, based on 8 article reviews
    Price from $9.99 to $1999.99
    bbsi digested lenticrisprv2 control vector - by Bioz Stars, 2020-02
    81/100 stars
      Buy from Supplier

    82
    Addgene inc crispr cas9 knockout plasmids
    Optimization of 2CT-CRISPR assay system for genome-scale screening a, Representative FACS plot of B2M expression in Mel624 cells on day 5 after transduction with <t>lentiCRISPRv2</t> lentivirus containing a pool of three sgRNAs targeting B2M . b–c, Cas9 disruption of MHC Class I antigen presentation/processing pathway genes reduces efficacy of T cell-mediated cytolysis. Timeline shows 12 hours of co-culture of ESO T cells with individual gene edited Mel624 cells at E:T ratio of 0.5. Live cell survival (%) was calculated from control cells unexposed to T cell selection. Each dot in the plot represents independent gene-specific CRISPR lentivirus infection replicate ( n = 3). Improvement in CRISPR edited cell yields at 60 h timepoint compared to 36 h after 2CT assay as shown in ( c) . All values are mean ± s.e.m. Data is representative of two independent experiments.
    Crispr Cas9 Knockout Plasmids, supplied by Addgene inc, used in various techniques. Bioz Stars score: 82/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/crispr cas9 knockout plasmids/product/Addgene inc
    Average 82 stars, based on 8 article reviews
    Price from $9.99 to $1999.99
    crispr cas9 knockout plasmids - by Bioz Stars, 2020-02
    82/100 stars
      Buy from Supplier

    80
    Addgene inc bsmbi digested lenticrisprv2 puromycin
    Optimization of 2CT-CRISPR assay system for genome-scale screening a, Representative FACS plot of B2M expression in Mel624 cells on day 5 after transduction with <t>lentiCRISPRv2</t> lentivirus containing a pool of three sgRNAs targeting B2M . b–c, Cas9 disruption of MHC Class I antigen presentation/processing pathway genes reduces efficacy of T cell-mediated cytolysis. Timeline shows 12 hours of co-culture of ESO T cells with individual gene edited Mel624 cells at E:T ratio of 0.5. Live cell survival (%) was calculated from control cells unexposed to T cell selection. Each dot in the plot represents independent gene-specific CRISPR lentivirus infection replicate ( n = 3). Improvement in CRISPR edited cell yields at 60 h timepoint compared to 36 h after 2CT assay as shown in ( c) . All values are mean ± s.e.m. Data is representative of two independent experiments.
    Bsmbi Digested Lenticrisprv2 Puromycin, supplied by Addgene inc, used in various techniques. Bioz Stars score: 80/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/bsmbi digested lenticrisprv2 puromycin/product/Addgene inc
    Average 80 stars, based on 8 article reviews
    Price from $9.99 to $1999.99
    bsmbi digested lenticrisprv2 puromycin - by Bioz Stars, 2020-02
    80/100 stars
      Buy from Supplier

    82
    Addgene inc lenticrisprv2 vector system
    Optimization of 2CT-CRISPR assay system for genome-scale screening a, Representative FACS plot of B2M expression in Mel624 cells on day 5 after transduction with <t>lentiCRISPRv2</t> lentivirus containing a pool of three sgRNAs targeting B2M . b–c, Cas9 disruption of MHC Class I antigen presentation/processing pathway genes reduces efficacy of T cell-mediated cytolysis. Timeline shows 12 hours of co-culture of ESO T cells with individual gene edited Mel624 cells at E:T ratio of 0.5. Live cell survival (%) was calculated from control cells unexposed to T cell selection. Each dot in the plot represents independent gene-specific CRISPR lentivirus infection replicate ( n = 3). Improvement in CRISPR edited cell yields at 60 h timepoint compared to 36 h after 2CT assay as shown in ( c) . All values are mean ± s.e.m. Data is representative of two independent experiments.
    Lenticrisprv2 Vector System, supplied by Addgene inc, used in various techniques. Bioz Stars score: 82/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/lenticrisprv2 vector system/product/Addgene inc
    Average 82 stars, based on 6 article reviews
    Price from $9.99 to $1999.99
    lenticrisprv2 vector system - by Bioz Stars, 2020-02
    82/100 stars
      Buy from Supplier

    87
    Addgene inc lenticrisprv2 transfer plasmid
    GFP-Tsg101 knock-in using CRISPR-Cas9 and HDR. A , knock-in strategy. Cells were transduced with <t>LentiCRISPRv2</t> to express Cas9 with sgRNA targeting the start of the Tsg101 gene and transfected with a donor template plasmid containing a puromycin resistance selection marker and a GFP tag flanked by HRs for the target site. Cas9 generates a DSB in the genomic DNA at the target site, and the donor template can be integrated into the DSB by HDR, resulting in GFP knock-in. Otherwise, the DSB is repaired by the more efficient process of NHEJ, leaving insertions or deletions that knock out Tsg101. Tsg101 knockout was lethal, which helped to select against cells that did not achieve the knock-in. B , Western blotting for Tsg101 in cell lysates of GFP-Tsg101 KI clonal lines and WT parental lines. The KI cell lines express GFP-Tsg101 at lower than WT levels and do not express untagged Tsg101. α-Tubulin is shown as a loading control.
    Lenticrisprv2 Transfer Plasmid, supplied by Addgene inc, used in various techniques. Bioz Stars score: 87/100, based on 12 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/lenticrisprv2 transfer plasmid/product/Addgene inc
    Average 87 stars, based on 12 article reviews
    Price from $9.99 to $1999.99
    lenticrisprv2 transfer plasmid - by Bioz Stars, 2020-02
    87/100 stars
      Buy from Supplier

    79
    Addgene inc lentivirus mediated crispr silencing lenticrisprv2
    GFP-Tsg101 knock-in using CRISPR-Cas9 and HDR. A , knock-in strategy. Cells were transduced with <t>LentiCRISPRv2</t> to express Cas9 with sgRNA targeting the start of the Tsg101 gene and transfected with a donor template plasmid containing a puromycin resistance selection marker and a GFP tag flanked by HRs for the target site. Cas9 generates a DSB in the genomic DNA at the target site, and the donor template can be integrated into the DSB by HDR, resulting in GFP knock-in. Otherwise, the DSB is repaired by the more efficient process of NHEJ, leaving insertions or deletions that knock out Tsg101. Tsg101 knockout was lethal, which helped to select against cells that did not achieve the knock-in. B , Western blotting for Tsg101 in cell lysates of GFP-Tsg101 KI clonal lines and WT parental lines. The KI cell lines express GFP-Tsg101 at lower than WT levels and do not express untagged Tsg101. α-Tubulin is shown as a loading control.
    Lentivirus Mediated Crispr Silencing Lenticrisprv2, supplied by Addgene inc, used in various techniques. Bioz Stars score: 79/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/lentivirus mediated crispr silencing lenticrisprv2/product/Addgene inc
    Average 79 stars, based on 4 article reviews
    Price from $9.99 to $1999.99
    lentivirus mediated crispr silencing lenticrisprv2 - by Bioz Stars, 2020-02
    79/100 stars
      Buy from Supplier

    84
    Addgene inc human gecko lentiviral grna library v2
    GFP-Tsg101 knock-in using CRISPR-Cas9 and HDR. A , knock-in strategy. Cells were transduced with <t>LentiCRISPRv2</t> to express Cas9 with sgRNA targeting the start of the Tsg101 gene and transfected with a donor template plasmid containing a puromycin resistance selection marker and a GFP tag flanked by HRs for the target site. Cas9 generates a DSB in the genomic DNA at the target site, and the donor template can be integrated into the DSB by HDR, resulting in GFP knock-in. Otherwise, the DSB is repaired by the more efficient process of NHEJ, leaving insertions or deletions that knock out Tsg101. Tsg101 knockout was lethal, which helped to select against cells that did not achieve the knock-in. B , Western blotting for Tsg101 in cell lysates of GFP-Tsg101 KI clonal lines and WT parental lines. The KI cell lines express GFP-Tsg101 at lower than WT levels and do not express untagged Tsg101. α-Tubulin is shown as a loading control.
    Human Gecko Lentiviral Grna Library V2, supplied by Addgene inc, used in various techniques. Bioz Stars score: 84/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/human gecko lentiviral grna library v2/product/Addgene inc
    Average 84 stars, based on 7 article reviews
    Price from $9.99 to $1999.99
    human gecko lentiviral grna library v2 - by Bioz Stars, 2020-02
    84/100 stars
      Buy from Supplier

    86
    Addgene inc lentivirus transfer vector lenticrisprv2
    GFP-Tsg101 knock-in using CRISPR-Cas9 and HDR. A , knock-in strategy. Cells were transduced with <t>LentiCRISPRv2</t> to express Cas9 with sgRNA targeting the start of the Tsg101 gene and transfected with a donor template plasmid containing a puromycin resistance selection marker and a GFP tag flanked by HRs for the target site. Cas9 generates a DSB in the genomic DNA at the target site, and the donor template can be integrated into the DSB by HDR, resulting in GFP knock-in. Otherwise, the DSB is repaired by the more efficient process of NHEJ, leaving insertions or deletions that knock out Tsg101. Tsg101 knockout was lethal, which helped to select against cells that did not achieve the knock-in. B , Western blotting for Tsg101 in cell lysates of GFP-Tsg101 KI clonal lines and WT parental lines. The KI cell lines express GFP-Tsg101 at lower than WT levels and do not express untagged Tsg101. α-Tubulin is shown as a loading control.
    Lentivirus Transfer Vector Lenticrisprv2, supplied by Addgene inc, used in various techniques. Bioz Stars score: 86/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/lentivirus transfer vector lenticrisprv2/product/Addgene inc
    Average 86 stars, based on 9 article reviews
    Price from $9.99 to $1999.99
    lentivirus transfer vector lenticrisprv2 - by Bioz Stars, 2020-02
    86/100 stars
      Buy from Supplier

    82
    Addgene inc guide rna cloning lenticrisprv2 vector
    GFP-Tsg101 knock-in using CRISPR-Cas9 and HDR. A , knock-in strategy. Cells were transduced with <t>LentiCRISPRv2</t> to express Cas9 with sgRNA targeting the start of the Tsg101 gene and transfected with a donor template plasmid containing a puromycin resistance selection marker and a GFP tag flanked by HRs for the target site. Cas9 generates a DSB in the genomic DNA at the target site, and the donor template can be integrated into the DSB by HDR, resulting in GFP knock-in. Otherwise, the DSB is repaired by the more efficient process of NHEJ, leaving insertions or deletions that knock out Tsg101. Tsg101 knockout was lethal, which helped to select against cells that did not achieve the knock-in. B , Western blotting for Tsg101 in cell lysates of GFP-Tsg101 KI clonal lines and WT parental lines. The KI cell lines express GFP-Tsg101 at lower than WT levels and do not express untagged Tsg101. α-Tubulin is shown as a loading control.
    Guide Rna Cloning Lenticrisprv2 Vector, supplied by Addgene inc, used in various techniques. Bioz Stars score: 82/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/guide rna cloning lenticrisprv2 vector/product/Addgene inc
    Average 82 stars, based on 5 article reviews
    Price from $9.99 to $1999.99
    guide rna cloning lenticrisprv2 vector - by Bioz Stars, 2020-02
    82/100 stars
      Buy from Supplier

    79
    Addgene inc cancer cells lenticrisprv2 lentiviral plasmid system
    GFP-Tsg101 knock-in using CRISPR-Cas9 and HDR. A , knock-in strategy. Cells were transduced with <t>LentiCRISPRv2</t> to express Cas9 with sgRNA targeting the start of the Tsg101 gene and transfected with a donor template plasmid containing a puromycin resistance selection marker and a GFP tag flanked by HRs for the target site. Cas9 generates a DSB in the genomic DNA at the target site, and the donor template can be integrated into the DSB by HDR, resulting in GFP knock-in. Otherwise, the DSB is repaired by the more efficient process of NHEJ, leaving insertions or deletions that knock out Tsg101. Tsg101 knockout was lethal, which helped to select against cells that did not achieve the knock-in. B , Western blotting for Tsg101 in cell lysates of GFP-Tsg101 KI clonal lines and WT parental lines. The KI cell lines express GFP-Tsg101 at lower than WT levels and do not express untagged Tsg101. α-Tubulin is shown as a loading control.
    Cancer Cells Lenticrisprv2 Lentiviral Plasmid System, supplied by Addgene inc, used in various techniques. Bioz Stars score: 79/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/cancer cells lenticrisprv2 lentiviral plasmid system/product/Addgene inc
    Average 79 stars, based on 5 article reviews
    Price from $9.99 to $1999.99
    cancer cells lenticrisprv2 lentiviral plasmid system - by Bioz Stars, 2020-02
    79/100 stars
      Buy from Supplier

    80
    Addgene inc lentivirus expressing crispr cas9 vector
    GFP-Tsg101 knock-in using CRISPR-Cas9 and HDR. A , knock-in strategy. Cells were transduced with <t>LentiCRISPRv2</t> to express Cas9 with sgRNA targeting the start of the Tsg101 gene and transfected with a donor template plasmid containing a puromycin resistance selection marker and a GFP tag flanked by HRs for the target site. Cas9 generates a DSB in the genomic DNA at the target site, and the donor template can be integrated into the DSB by HDR, resulting in GFP knock-in. Otherwise, the DSB is repaired by the more efficient process of NHEJ, leaving insertions or deletions that knock out Tsg101. Tsg101 knockout was lethal, which helped to select against cells that did not achieve the knock-in. B , Western blotting for Tsg101 in cell lysates of GFP-Tsg101 KI clonal lines and WT parental lines. The KI cell lines express GFP-Tsg101 at lower than WT levels and do not express untagged Tsg101. α-Tubulin is shown as a loading control.
    Lentivirus Expressing Crispr Cas9 Vector, supplied by Addgene inc, used in various techniques. Bioz Stars score: 80/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/lentivirus expressing crispr cas9 vector/product/Addgene inc
    Average 80 stars, based on 4 article reviews
    Price from $9.99 to $1999.99
    lentivirus expressing crispr cas9 vector - by Bioz Stars, 2020-02
    80/100 stars
      Buy from Supplier

    80
    Addgene inc cloning crispr cas9 venus plasmids ecpv lenticrisprv2 plasmid
    GFP-Tsg101 knock-in using CRISPR-Cas9 and HDR. A , knock-in strategy. Cells were transduced with <t>LentiCRISPRv2</t> to express Cas9 with sgRNA targeting the start of the Tsg101 gene and transfected with a donor template plasmid containing a puromycin resistance selection marker and a GFP tag flanked by HRs for the target site. Cas9 generates a DSB in the genomic DNA at the target site, and the donor template can be integrated into the DSB by HDR, resulting in GFP knock-in. Otherwise, the DSB is repaired by the more efficient process of NHEJ, leaving insertions or deletions that knock out Tsg101. Tsg101 knockout was lethal, which helped to select against cells that did not achieve the knock-in. B , Western blotting for Tsg101 in cell lysates of GFP-Tsg101 KI clonal lines and WT parental lines. The KI cell lines express GFP-Tsg101 at lower than WT levels and do not express untagged Tsg101. α-Tubulin is shown as a loading control.
    Cloning Crispr Cas9 Venus Plasmids Ecpv Lenticrisprv2 Plasmid, supplied by Addgene inc, used in various techniques. Bioz Stars score: 80/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/cloning crispr cas9 venus plasmids ecpv lenticrisprv2 plasmid/product/Addgene inc
    Average 80 stars, based on 6 article reviews
    Price from $9.99 to $1999.99
    cloning crispr cas9 venus plasmids ecpv lenticrisprv2 plasmid - by Bioz Stars, 2020-02
    80/100 stars
      Buy from Supplier

    87
    Addgene inc cas9 expressing lentiviral transfer vector
    PGRMC1 knockout using a <t>CRISPR/Cas9</t> approach. A. Schematic of the PGRMC1 sgRNA/Cas9-expressing <t>lentiviral</t> constructs for knocking out PGRMC1. Gray areas show the three candidate sgRNA sequences in the exon-1 of the PGRMC1 gene, of which two were used for the CRISPR/Cas9 constructs. B. A representative DNA gel of control and PGRMC1 knockout (clones 38 and 207) NSC34 cells verifying the Cas9 cleavage of the genomic DNA. Control refers to the NSC34 cells transfected with the control vector expressing Cas9 but not an sgRNA. C. Western blotting detection of PGRMC1 in control and PGRMC1 knockout (clones 38 and 207) NSC34 cells.
    Cas9 Expressing Lentiviral Transfer Vector, supplied by Addgene inc, used in various techniques. Bioz Stars score: 87/100, based on 17 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/cas9 expressing lentiviral transfer vector/product/Addgene inc
    Average 87 stars, based on 17 article reviews
    Price from $9.99 to $1999.99
    cas9 expressing lentiviral transfer vector - by Bioz Stars, 2020-02
    87/100 stars
      Buy from Supplier

    Image Search Results


    GFP-Tsg101 knock-in using CRISPR-Cas9 and HDR. A , knock-in strategy. Cells were transduced with LentiCRISPRv2 to express Cas9 with sgRNA targeting the start of the Tsg101 gene and transfected with a donor template plasmid containing a puromycin resistance selection marker and a GFP tag flanked by HRs for the target site. Cas9 generates a DSB in the genomic DNA at the target site, and the donor template can be integrated into the DSB by HDR, resulting in GFP knock-in. Otherwise, the DSB is repaired by the more efficient process of NHEJ, leaving insertions or deletions that knock out Tsg101. Tsg101 knockout was lethal, which helped to select against cells that did not achieve the knock-in. B , Western blotting for Tsg101 in cell lysates of GFP-Tsg101 KI clonal lines and WT parental lines. The KI cell lines express GFP-Tsg101 at lower than WT levels and do not express untagged Tsg101. α-Tubulin is shown as a loading control.

    Journal: The Journal of Biological Chemistry

    Article Title: Genomic tagging of endogenous human ESCRT-I complex preserves ESCRT-mediated membrane-remodeling functions

    doi: 10.1074/jbc.RA119.009372

    Figure Lengend Snippet: GFP-Tsg101 knock-in using CRISPR-Cas9 and HDR. A , knock-in strategy. Cells were transduced with LentiCRISPRv2 to express Cas9 with sgRNA targeting the start of the Tsg101 gene and transfected with a donor template plasmid containing a puromycin resistance selection marker and a GFP tag flanked by HRs for the target site. Cas9 generates a DSB in the genomic DNA at the target site, and the donor template can be integrated into the DSB by HDR, resulting in GFP knock-in. Otherwise, the DSB is repaired by the more efficient process of NHEJ, leaving insertions or deletions that knock out Tsg101. Tsg101 knockout was lethal, which helped to select against cells that did not achieve the knock-in. B , Western blotting for Tsg101 in cell lysates of GFP-Tsg101 KI clonal lines and WT parental lines. The KI cell lines express GFP-Tsg101 at lower than WT levels and do not express untagged Tsg101. α-Tubulin is shown as a loading control.

    Article Snippet: We thank Dr. Feng Zhang for the gift of LentiCRISPRv2 (Addgene plasmid 52961), Dr. Heinrich Leonhardt for the gift of pCANTD-EGFP, Dr. David Sabatini for the gift of pLJM1-EGFP (Addgene plasmid 19319), Dr. Didier Trono for the gift of psPAX2 (Addgene plasmid 12260), and Dr. Xuedong Liu for the gift of VSV-G.

    Techniques: Knock-In, CRISPR, Transduction, Transfection, Plasmid Preparation, Selection, Marker, Non-Homologous End Joining, Knock-Out, Western Blot

    GABA A β1-3 subunits are necessary for inhibitory transmission A) Diagram of the vector constructs used to express the chained sgRNAs (in pink) for β1, β2, and β3 and Cas9 (in yellow). CBh: promoter; NLS: nuclear localization sequence; 2A: cleavage peptide; U6: promoter; hUbC: promoter. B) Western blot of rat dissociated hippocampal cultures infected with lentiviruses for both Cas9 and sgRNA (CRISPR lanes) or cultures transfected with lentivirus only for Cas9 (Control lanes). Lysates were probed for expression of β1, β2/3, and actin (n=4 replicates). C) Representative image of our organotypic hippocampal slice culture preparation with a zoomed in image of a transfected cell. D) Representative evoked IPSC traces of an untransfected cell (in black) and cell transfected with the sgRNAs for β1-3 and Cas9 (in green). E) Varying absolute amplitudes were observed in control untransfected cells while transfected cells displayed no inhibitory current (***p = 0.0002, n = 18) F) .

    Journal: Neuron

    Article Title: The GABAA receptor β subunit is required for inhibitory transmission

    doi: 10.1016/j.neuron.2018.03.046

    Figure Lengend Snippet: GABA A β1-3 subunits are necessary for inhibitory transmission A) Diagram of the vector constructs used to express the chained sgRNAs (in pink) for β1, β2, and β3 and Cas9 (in yellow). CBh: promoter; NLS: nuclear localization sequence; 2A: cleavage peptide; U6: promoter; hUbC: promoter. B) Western blot of rat dissociated hippocampal cultures infected with lentiviruses for both Cas9 and sgRNA (CRISPR lanes) or cultures transfected with lentivirus only for Cas9 (Control lanes). Lysates were probed for expression of β1, β2/3, and actin (n=4 replicates). C) Representative image of our organotypic hippocampal slice culture preparation with a zoomed in image of a transfected cell. D) Representative evoked IPSC traces of an untransfected cell (in black) and cell transfected with the sgRNAs for β1-3 and Cas9 (in green). E) Varying absolute amplitudes were observed in control untransfected cells while transfected cells displayed no inhibitory current (***p = 0.0002, n = 18) F) .

    Article Snippet: The human codon-optimized Cas9 and chimeric sgRNA expression plasmid (pX458) and lentiviral plasmid for expression of Cas9 (lentiCRISPRv.2) were developed by the Zhang lab and obtained from Addgene (plasmid #48138 and #52961) ( ; ).

    Techniques: Transmission Assay, Plasmid Preparation, Construct, Sequencing, Western Blot, Infection, CRISPR, Transfection, Expressing

    MVM associates with artificially-engineered sites of DNA damage. ( A ) Murine A9 fibroblasts were infected with MVM at an MOI of 10 for 18 hr. Cells were sensitized with Hoechst 33342 solution prior to irradiation in selected regions of interest with a 405 nanometer laser (top 3 panels), and were compared with un-irradiated cells (bottom panel). Cells were fixed and analyzed for the localization of DNA damaged sites (detected by green γ-H2AX staining), with that of MVM NS1 protein ( red ). The irrelevant transcription factor NR5A2 ( red ) was used as a negative control as this marker that does not colocalize with APAR bodies (see Figure 1 ). The nuclear periphery is demarcated with dotted white lines and DAPI staining. ( B ) Schematic of γ-H2AX binding to the cellular genome on chromosome 9 (top panel) compared with MVM-interaction sites (bottom panel) in parasynchronized murine A9 cells infected with MVM (MOI 5) for 16 hr. The site where guide RNAs were designed for DDR induction is shown as a red rectangle and labelled ‘CRISPR/Cas9’. Focused V3C-qPCR assays were performed in A9 cells constitutively expressing LentiCRISPRv2 which were transiently transfected with guide RNAs (nontargeting, labelled as ‘CTRL’ or specific to the 9qE1 site, labelled as ‘TGT’), and infected with MVM at an MOI for 5 for 16 hr. The spatial association of MVM with cellular sites were determined with Taqman probes on ( C ), the MVM genome, and reciprocal interaction with the Taqman probe on ( D ), the 9qE1 break site. ( E ) MVM association with Chr19 VAD at 19qA was tested by focused Taqman qPCR. NS1 occupancy at ( F ), the 9qE1 break site and ( G ), the 19qA VAD was assayed by ChIP-qPCR in cells transfected with non-targeting (CTRL) and specific (TGT) guide RNAs. Background levels were determined using IgG pulldowns. qPCR data are presented as mean ± SEM of three independent experiments.

    Journal: eLife

    Article Title: Parvovirus minute virus of mice interacts with sites of cellular DNA damage to establish and amplify its lytic infection

    doi: 10.7554/eLife.37750

    Figure Lengend Snippet: MVM associates with artificially-engineered sites of DNA damage. ( A ) Murine A9 fibroblasts were infected with MVM at an MOI of 10 for 18 hr. Cells were sensitized with Hoechst 33342 solution prior to irradiation in selected regions of interest with a 405 nanometer laser (top 3 panels), and were compared with un-irradiated cells (bottom panel). Cells were fixed and analyzed for the localization of DNA damaged sites (detected by green γ-H2AX staining), with that of MVM NS1 protein ( red ). The irrelevant transcription factor NR5A2 ( red ) was used as a negative control as this marker that does not colocalize with APAR bodies (see Figure 1 ). The nuclear periphery is demarcated with dotted white lines and DAPI staining. ( B ) Schematic of γ-H2AX binding to the cellular genome on chromosome 9 (top panel) compared with MVM-interaction sites (bottom panel) in parasynchronized murine A9 cells infected with MVM (MOI 5) for 16 hr. The site where guide RNAs were designed for DDR induction is shown as a red rectangle and labelled ‘CRISPR/Cas9’. Focused V3C-qPCR assays were performed in A9 cells constitutively expressing LentiCRISPRv2 which were transiently transfected with guide RNAs (nontargeting, labelled as ‘CTRL’ or specific to the 9qE1 site, labelled as ‘TGT’), and infected with MVM at an MOI for 5 for 16 hr. The spatial association of MVM with cellular sites were determined with Taqman probes on ( C ), the MVM genome, and reciprocal interaction with the Taqman probe on ( D ), the 9qE1 break site. ( E ) MVM association with Chr19 VAD at 19qA was tested by focused Taqman qPCR. NS1 occupancy at ( F ), the 9qE1 break site and ( G ), the 19qA VAD was assayed by ChIP-qPCR in cells transfected with non-targeting (CTRL) and specific (TGT) guide RNAs. Background levels were determined using IgG pulldowns. qPCR data are presented as mean ± SEM of three independent experiments.

    Article Snippet: LentiCRISPRv2 A9 cells LentiCRISPRv2 plasmid was obtained from Addgene (plasmid# 52961, [ ]) and pseudotyped viruses were generated in 1 × 106 293 T cells transfected with 1 µg of LentiCRISPRv2, 1 µg of HIV Gag/Pol and 1 µg VSV-G proteins using Lipo293D (SignaGen).

    Techniques: Infection, Irradiation, Staining, Negative Control, Marker, Binding Assay, CRISPR, Real-time Polymerase Chain Reaction, Expressing, Transfection, Chromatin Immunoprecipitation

    Optimization of 2CT-CRISPR assay system for genome-scale screening a, Representative FACS plot of B2M expression in Mel624 cells on day 5 after transduction with lentiCRISPRv2 lentivirus containing a pool of three sgRNAs targeting B2M . b–c, Cas9 disruption of MHC Class I antigen presentation/processing pathway genes reduces efficacy of T cell-mediated cytolysis. Timeline shows 12 hours of co-culture of ESO T cells with individual gene edited Mel624 cells at E:T ratio of 0.5. Live cell survival (%) was calculated from control cells unexposed to T cell selection. Each dot in the plot represents independent gene-specific CRISPR lentivirus infection replicate ( n = 3). Improvement in CRISPR edited cell yields at 60 h timepoint compared to 36 h after 2CT assay as shown in ( c) . All values are mean ± s.e.m. Data is representative of two independent experiments.

    Journal: Nature

    Article Title: Identification of essential genes for cancer immunotherapy

    doi: 10.1038/nature23477

    Figure Lengend Snippet: Optimization of 2CT-CRISPR assay system for genome-scale screening a, Representative FACS plot of B2M expression in Mel624 cells on day 5 after transduction with lentiCRISPRv2 lentivirus containing a pool of three sgRNAs targeting B2M . b–c, Cas9 disruption of MHC Class I antigen presentation/processing pathway genes reduces efficacy of T cell-mediated cytolysis. Timeline shows 12 hours of co-culture of ESO T cells with individual gene edited Mel624 cells at E:T ratio of 0.5. Live cell survival (%) was calculated from control cells unexposed to T cell selection. Each dot in the plot represents independent gene-specific CRISPR lentivirus infection replicate ( n = 3). Improvement in CRISPR edited cell yields at 60 h timepoint compared to 36 h after 2CT assay as shown in ( c) . All values are mean ± s.e.m. Data is representative of two independent experiments.

    Article Snippet: For each flask, 4 mL of OptiMEM was mixed with 200 μL of Plus reagent, 20 μg of lentiCRISPRv2 plasmid or pooled plasmid human GeCKOv2 (Genome-scale CRISPR Knock-Out) library, 15 μg psPAX2 (Addgene, Cambridge, MA) and 10 μg pMD2.G (Addgene).

    Techniques: CRISPR, FACS, Expressing, Transduction, Co-Culture Assay, Selection, Infection

    DNA, RNA, and Protein Expression Analysis after Infection with LentiCRISPRv2 (A) Histograms reporting the genomic analysis by real-time PCR of the duplicated sequence. Transduced cells present reduction of the duplication in respect to untreated cells (UT) set to 1. Wild-type cells were analyzed as positive control. (B) RT-PCR was performed with primers designed on the exon 1 and 4. One microliter of the displayed PCR was loaded on a high-sensitivity DNA chip and run on the Bioanalyser 2100 (Agilent). Quantification of the peak obtained was performed calculating the ratio of the area of the normal transcript and the sum of the area of normal and duplicated transcript multiplied for 100 and normalized by setting the untreated (UT) as one. *p

    Journal: Molecular Therapy. Nucleic Acids

    Article Title: Correction of the Exon 2 Duplication in DMD Myoblasts by a Single CRISPR/Cas9 System

    doi: 10.1016/j.omtn.2017.02.004

    Figure Lengend Snippet: DNA, RNA, and Protein Expression Analysis after Infection with LentiCRISPRv2 (A) Histograms reporting the genomic analysis by real-time PCR of the duplicated sequence. Transduced cells present reduction of the duplication in respect to untreated cells (UT) set to 1. Wild-type cells were analyzed as positive control. (B) RT-PCR was performed with primers designed on the exon 1 and 4. One microliter of the displayed PCR was loaded on a high-sensitivity DNA chip and run on the Bioanalyser 2100 (Agilent). Quantification of the peak obtained was performed calculating the ratio of the area of the normal transcript and the sum of the area of normal and duplicated transcript multiplied for 100 and normalized by setting the untreated (UT) as one. *p

    Article Snippet: We did not select the infected cells by antibiotics resistance as the immortalization process takes advantage of two lentiviruses and one shares the puromycin resistance also present in the LentiCRISPRv2 vector.

    Techniques: Expressing, Infection, Real-time Polymerase Chain Reaction, Sequencing, Positive Control, Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction, Chromatin Immunoprecipitation

    Generation of the miR-26a-knockout line in HeLa cells using the CRISPR-Cas9 system. ( a ). ( b ) The sequencing results showed that the two sgRNAs were appropriately inserted into the lentiCRISPRv2 vector. ( c ) The sequencing data revealed that a 28-bp deletion in the target site was introduced by the CRISPR-Cas9 system.

    Journal: Scientific Reports

    Article Title: Label-free Quantitative Analysis of Protein Expression Alterations in miR-26a-Knockout HeLa Cells using SWATH-MS Technology

    doi: 10.1038/s41598-018-34904-8

    Figure Lengend Snippet: Generation of the miR-26a-knockout line in HeLa cells using the CRISPR-Cas9 system. ( a ). ( b ) The sequencing results showed that the two sgRNAs were appropriately inserted into the lentiCRISPRv2 vector. ( c ) The sequencing data revealed that a 28-bp deletion in the target site was introduced by the CRISPR-Cas9 system.

    Article Snippet: To produce the lentivirus, HEK 293 T cells seeded in 100-mm plates were cotransfected with 4.0 μg of lentiCRISPRv2 plasmids, 3.0 μg of psPAX2 (Addgene plasmid # 12260), and 1.0 μg of pMD2.G plasmids (Addgene plasmid #12259) using polyethyleneimine (Polysciences Inc., USA), according to the manufacturer’s instructions.

    Techniques: Knock-Out, CRISPR, Sequencing, Plasmid Preparation

    TRIM5alpha, IFITM1 and Tetherin are additional ISGs that contribute to the IFN block. ( A ) KO efficiencies in lentiCRISPRv2-edited THP-1 cells as determined by ICE analysis (left) or flow cytometry (right – pretreated with 1000 U/mL uIFN). ( B ) THP-1 cell pools edited for gene targets of interest were created by transducing wild type THP-1 cells with lentiCRISPRv2 sgRNA constructs (two different sgRNAs - for STAT1, STAT2, IRF9, MxB, TRIM5alpha and Tetherin, and three for IFITM1), selected for 2 weeks to allow gene knockout (see Figure 4—source data 1 Supplemental file 1 for analysis of gene knockout efficiency) and infected with HIV-1 LAI with and without IFNα pretreatment in triplicate (white bars = no IFNα, gray bars =+IFNα). The percentage of cells expressing HIV p24gag was assayed 2 days post-infection by intracellular staining and flow cytometry. NTC n = 9. MxB_2 n = 6. All other pools n = 3. ( C ) The Fold Inhibition (%p24 +cells without IFN/%p24 +cells with IFNα) is shown for each KO pool. Control (NTC) = gray; IFN pathway genes = magenta. MxB, TRIM5alpha, IFITM1 and Tetherin = Cyan. Cells pools with significantly reduced Fold Inhibition as compared to the NTC pools *p

    Journal: eLife

    Article Title: A virus-packageable CRISPR screen identifies host factors mediating interferon inhibition of HIV

    doi: 10.7554/eLife.39823

    Figure Lengend Snippet: TRIM5alpha, IFITM1 and Tetherin are additional ISGs that contribute to the IFN block. ( A ) KO efficiencies in lentiCRISPRv2-edited THP-1 cells as determined by ICE analysis (left) or flow cytometry (right – pretreated with 1000 U/mL uIFN). ( B ) THP-1 cell pools edited for gene targets of interest were created by transducing wild type THP-1 cells with lentiCRISPRv2 sgRNA constructs (two different sgRNAs - for STAT1, STAT2, IRF9, MxB, TRIM5alpha and Tetherin, and three for IFITM1), selected for 2 weeks to allow gene knockout (see Figure 4—source data 1 Supplemental file 1 for analysis of gene knockout efficiency) and infected with HIV-1 LAI with and without IFNα pretreatment in triplicate (white bars = no IFNα, gray bars =+IFNα). The percentage of cells expressing HIV p24gag was assayed 2 days post-infection by intracellular staining and flow cytometry. NTC n = 9. MxB_2 n = 6. All other pools n = 3. ( C ) The Fold Inhibition (%p24 +cells without IFN/%p24 +cells with IFNα) is shown for each KO pool. Control (NTC) = gray; IFN pathway genes = magenta. MxB, TRIM5alpha, IFITM1 and Tetherin = Cyan. Cells pools with significantly reduced Fold Inhibition as compared to the NTC pools *p

    Article Snippet: Plasmids lentiCRISPRv2 plasmid was a gift from Feng Zhang (Addgene #52961). pMD2.G and psPAX2 were gifts from Didier Trono (Addgene #12259/12260). lentiCRISPRv2 constructs targeting genes of interest were cloned into BsmBI-digested lentiCRISPRv2 by annealing complementary oligos ( ) with overhangs that allow directional cloning into lentiCRISPRv2.

    Techniques: Blocking Assay, Flow Cytometry, Cytometry, Construct, Gene Knockout, Infection, Expressing, Staining, Inhibition

    MxB is a dominant, early-acting ISG whose activity is masked by other ISGs when HIV entry is mediated by VSV-G. ( A ) Clonal MxB-KO THP-1 lines generated by transducing with an MxB-targeting sgRNA/lentiCRISPRv2 construct, selection and single-cell sorting. Western blot for MxB expression with and without IFNα stimulation overnight is shown for wild type (wt) THP-1 cells; MxB-KO clones were all IFNα treated overnight. Note: the lower molecular weight band in some lanes results from initiation at an internal Met codon that would not be predicted to have anti-HIV activity ( Goujon et al., 2015 ; Matreyek et al., 2014 ). ( B ) Nine individual clonal THP-1 lines (white/gray bars) along with five clonal THP-1 MxB-KO lines (pink bars) were pre-treated with IFNα overnight and infected with wt HIV-1 LAI . The percentage of cells expressing HIV p24gag was assayed 2 days post-infection by intracellular staining and flow cytometry (n = 3). Light bars = no IFN; Dark bars = overnight IFNα treatment prior to infection. ( C ) The Fold Inhibition (%p24 +cells without IFN/%p24 +cells with IFNα) calculated for each clonal line for wt HIV-1 LAI infections from the data in Panel B. Controls = gray; MxB-KO = magenta. Dotted line at a Fold Inhibition of 1 = no IFN inhibition. ( D ) Individual clonal control THP-1 lines (gray) along with MxB-KO clonal lines (magenta) were infected with VSV-G pseudotyped HIV both with and without IFNα pretreatment (n = 3). Fold Inhibition was calculated as in C. Dotted line: Fold Inhibition of 1 = no IFN inhibition. *p = 0.0014 (unpaired t test). ( E ) The PIKA HIV screen was performed in triplicate in ZAP-KO THP-1 cells. Y-Axis: IFN induction as determined by Differential Expression Analysis of microarray data in THP-1 cells (log 2 FoldChange). X-Axis: MAGeCK Gene Scores for Top 25 Hits. Magenta: IFN pathway genes (IFNAR1, STAT1, STAT2, IRF9). Cyan: highly-IFN induced, high-scoring candidate Hits. White: non-IFN induced genes including ZAP, N4BP1, and SLC35A2. High-scoring genes with no information on IFN induction in THP-1s are plotted as IFN DE log 2 FC = 0 but shown in Cyan with a gray outline. 10.7554/eLife.39823.014 MAGeCK Gene Analysis (Positive) of ZAP-KO THP-1 PIKA HIV HIV-1 LAI /VSVG Screen. pos|score sort: id. num. pos|score. pos|p-value. pos|fdr. pos|rank. pos|goodsgrna. pos|lfc. pos|score(-log10).

    Journal: eLife

    Article Title: A virus-packageable CRISPR screen identifies host factors mediating interferon inhibition of HIV

    doi: 10.7554/eLife.39823

    Figure Lengend Snippet: MxB is a dominant, early-acting ISG whose activity is masked by other ISGs when HIV entry is mediated by VSV-G. ( A ) Clonal MxB-KO THP-1 lines generated by transducing with an MxB-targeting sgRNA/lentiCRISPRv2 construct, selection and single-cell sorting. Western blot for MxB expression with and without IFNα stimulation overnight is shown for wild type (wt) THP-1 cells; MxB-KO clones were all IFNα treated overnight. Note: the lower molecular weight band in some lanes results from initiation at an internal Met codon that would not be predicted to have anti-HIV activity ( Goujon et al., 2015 ; Matreyek et al., 2014 ). ( B ) Nine individual clonal THP-1 lines (white/gray bars) along with five clonal THP-1 MxB-KO lines (pink bars) were pre-treated with IFNα overnight and infected with wt HIV-1 LAI . The percentage of cells expressing HIV p24gag was assayed 2 days post-infection by intracellular staining and flow cytometry (n = 3). Light bars = no IFN; Dark bars = overnight IFNα treatment prior to infection. ( C ) The Fold Inhibition (%p24 +cells without IFN/%p24 +cells with IFNα) calculated for each clonal line for wt HIV-1 LAI infections from the data in Panel B. Controls = gray; MxB-KO = magenta. Dotted line at a Fold Inhibition of 1 = no IFN inhibition. ( D ) Individual clonal control THP-1 lines (gray) along with MxB-KO clonal lines (magenta) were infected with VSV-G pseudotyped HIV both with and without IFNα pretreatment (n = 3). Fold Inhibition was calculated as in C. Dotted line: Fold Inhibition of 1 = no IFN inhibition. *p = 0.0014 (unpaired t test). ( E ) The PIKA HIV screen was performed in triplicate in ZAP-KO THP-1 cells. Y-Axis: IFN induction as determined by Differential Expression Analysis of microarray data in THP-1 cells (log 2 FoldChange). X-Axis: MAGeCK Gene Scores for Top 25 Hits. Magenta: IFN pathway genes (IFNAR1, STAT1, STAT2, IRF9). Cyan: highly-IFN induced, high-scoring candidate Hits. White: non-IFN induced genes including ZAP, N4BP1, and SLC35A2. High-scoring genes with no information on IFN induction in THP-1s are plotted as IFN DE log 2 FC = 0 but shown in Cyan with a gray outline. 10.7554/eLife.39823.014 MAGeCK Gene Analysis (Positive) of ZAP-KO THP-1 PIKA HIV HIV-1 LAI /VSVG Screen. pos|score sort: id. num. pos|score. pos|p-value. pos|fdr. pos|rank. pos|goodsgrna. pos|lfc. pos|score(-log10).

    Article Snippet: Plasmids lentiCRISPRv2 plasmid was a gift from Feng Zhang (Addgene #52961). pMD2.G and psPAX2 were gifts from Didier Trono (Addgene #12259/12260). lentiCRISPRv2 constructs targeting genes of interest were cloned into BsmBI-digested lentiCRISPRv2 by annealing complementary oligos ( ) with overhangs that allow directional cloning into lentiCRISPRv2.

    Techniques: Activity Assay, Generated, Construct, Selection, FACS, Western Blot, Expressing, Clone Assay, Molecular Weight, Infection, Staining, Flow Cytometry, Cytometry, Inhibition, Microarray

    Knockout of uPAR by CRISPR/Cas9 system. (A) The map of lentiCRISPRv2 vector. (B) The locations and sequences of two sgRNAs of uPAR. (C) The protein expression levels of uPAR were examined by Western blot, and vinculin was used as loading control. The genomic DNA of cells was amplified and sequenced by the designed primers. The sequencing comparison and original data of HCT8/T (D) and KB V200 (E) cells are shown.

    Journal: Frontiers in Oncology

    Article Title: Targeting uPAR by CRISPR/Cas9 System Attenuates Cancer Malignancy and Multidrug Resistance

    doi: 10.3389/fonc.2019.00080

    Figure Lengend Snippet: Knockout of uPAR by CRISPR/Cas9 system. (A) The map of lentiCRISPRv2 vector. (B) The locations and sequences of two sgRNAs of uPAR. (C) The protein expression levels of uPAR were examined by Western blot, and vinculin was used as loading control. The genomic DNA of cells was amplified and sequenced by the designed primers. The sequencing comparison and original data of HCT8/T (D) and KB V200 (E) cells are shown.

    Article Snippet: Vector Generation, Lentivirus Production, and Transduction LentiCRISPRv2 vector (from Addgene #52961) was digested with BsmBI and ligated with annealed oligonucleotides (uPAR-sg1-F: 5′-CACCGGACCAACGGGGATTGCCGTG-3′, uPAR-sg1-R: 5′-AA-ACCACGGCAATCCCCGTTGGTCC-3′; uPAR-sg2-F: 5′-CACCGGGACCACGATCGTGCGCTTG-3′, uPAR-sg2-R: 5′-AAACCAAGCGCACGATCGTGGTCCC-3′).

    Techniques: Knock-Out, CRISPR, Plasmid Preparation, Expressing, Western Blot, Amplification, Sequencing

    MGP depletion stimulates osteoclast differentiation and activity. (A) Efficiency of MGP knockout in BMMs using a CRISPR-Cas9 system. (B) Knockout of MGP leads to an increase in the mRNA levels of osteoclastic markers. BMMs were induced with 30 ng/ml

    Journal: Molecular and Cellular Biology

    Article Title: Unexpected Role of Matrix Gla Protein in Osteoclasts: Inhibiting Osteoclast Differentiation and Bone Resorption

    doi: 10.1128/MCB.00012-19

    Figure Lengend Snippet: MGP depletion stimulates osteoclast differentiation and activity. (A) Efficiency of MGP knockout in BMMs using a CRISPR-Cas9 system. (B) Knockout of MGP leads to an increase in the mRNA levels of osteoclastic markers. BMMs were induced with 30 ng/ml

    Article Snippet: The lentivirus system for CRISPR-Cas9 includes LentiCRISPRv2 backbone vector (Addgene, 52961), packaging plasmids psPAX2 (Addgene, 12260), and pVSVg (Addgene, 8454). sgRNA oligonucleotides were annealed and ligated into the LentiCRISPRv2 vector, generating plasmids that expressed a gRNA targeting MGP or a nontargeting sgRNA, respectively.

    Techniques: Activity Assay, Knock-Out, CRISPR

    GFP-Tsg101 knock-in using CRISPR-Cas9 and HDR. A , knock-in strategy. Cells were transduced with LentiCRISPRv2 to express Cas9 with sgRNA targeting the start of the Tsg101 gene and transfected with a donor template plasmid containing a puromycin resistance selection marker and a GFP tag flanked by HRs for the target site. Cas9 generates a DSB in the genomic DNA at the target site, and the donor template can be integrated into the DSB by HDR, resulting in GFP knock-in. Otherwise, the DSB is repaired by the more efficient process of NHEJ, leaving insertions or deletions that knock out Tsg101. Tsg101 knockout was lethal, which helped to select against cells that did not achieve the knock-in. B , Western blotting for Tsg101 in cell lysates of GFP-Tsg101 KI clonal lines and WT parental lines. The KI cell lines express GFP-Tsg101 at lower than WT levels and do not express untagged Tsg101. α-Tubulin is shown as a loading control.

    Journal: The Journal of Biological Chemistry

    Article Title: Genomic tagging of endogenous human ESCRT-I complex preserves ESCRT-mediated membrane-remodeling functions

    doi: 10.1074/jbc.RA119.009372

    Figure Lengend Snippet: GFP-Tsg101 knock-in using CRISPR-Cas9 and HDR. A , knock-in strategy. Cells were transduced with LentiCRISPRv2 to express Cas9 with sgRNA targeting the start of the Tsg101 gene and transfected with a donor template plasmid containing a puromycin resistance selection marker and a GFP tag flanked by HRs for the target site. Cas9 generates a DSB in the genomic DNA at the target site, and the donor template can be integrated into the DSB by HDR, resulting in GFP knock-in. Otherwise, the DSB is repaired by the more efficient process of NHEJ, leaving insertions or deletions that knock out Tsg101. Tsg101 knockout was lethal, which helped to select against cells that did not achieve the knock-in. B , Western blotting for Tsg101 in cell lysates of GFP-Tsg101 KI clonal lines and WT parental lines. The KI cell lines express GFP-Tsg101 at lower than WT levels and do not express untagged Tsg101. α-Tubulin is shown as a loading control.

    Article Snippet: The LentiCRISPRv2 transfer plasmid was a gift from Feng Zhang (Addgene plasmid 52961).

    Techniques: Knock-In, CRISPR, Transduction, Transfection, Plasmid Preparation, Selection, Marker, Non-Homologous End Joining, Knock-Out, Western Blot

    PGRMC1 knockout using a CRISPR/Cas9 approach. A. Schematic of the PGRMC1 sgRNA/Cas9-expressing lentiviral constructs for knocking out PGRMC1. Gray areas show the three candidate sgRNA sequences in the exon-1 of the PGRMC1 gene, of which two were used for the CRISPR/Cas9 constructs. B. A representative DNA gel of control and PGRMC1 knockout (clones 38 and 207) NSC34 cells verifying the Cas9 cleavage of the genomic DNA. Control refers to the NSC34 cells transfected with the control vector expressing Cas9 but not an sgRNA. C. Western blotting detection of PGRMC1 in control and PGRMC1 knockout (clones 38 and 207) NSC34 cells.

    Journal: EBioMedicine

    Article Title: The Sigma-2 Receptor and Progesterone Receptor Membrane Component 1 are Different Binding Sites Derived From Independent Genes

    doi: 10.1016/j.ebiom.2015.10.017

    Figure Lengend Snippet: PGRMC1 knockout using a CRISPR/Cas9 approach. A. Schematic of the PGRMC1 sgRNA/Cas9-expressing lentiviral constructs for knocking out PGRMC1. Gray areas show the three candidate sgRNA sequences in the exon-1 of the PGRMC1 gene, of which two were used for the CRISPR/Cas9 constructs. B. A representative DNA gel of control and PGRMC1 knockout (clones 38 and 207) NSC34 cells verifying the Cas9 cleavage of the genomic DNA. Control refers to the NSC34 cells transfected with the control vector expressing Cas9 but not an sgRNA. C. Western blotting detection of PGRMC1 in control and PGRMC1 knockout (clones 38 and 207) NSC34 cells.

    Article Snippet: 2.3 Generation of CRISPR/Cas9 Constructs for PGRMC1 Knockout For knocking out PGRMC1 using the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology, sgRNAs targeting PGRMC1 exon-1 were cloned into a Cas9-expressing lentiviral transfer vector (lentiCRISPRv2, Cat No. 52961, Addgene, Cambridge, MA) following the methods of the Feng Zhang laboratory ( ).

    Techniques: Knock-Out, CRISPR, Expressing, Construct, Transfection, Plasmid Preparation, Western Blot