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guide rna pairs  (Synthego Inc)

 
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    Synthego Inc guide rna pairs
    A. Schematic of β-catenin interaction with E-cadherin at the plasma membrane. B. Immunoblot analysis of total E-cadherin and β-catenin protein abundance in iPSCs. N = 3 clonal lines each were used, and Tubulin serves as loading control. C. Representative flow plots showing surface E-cadherin in control (dark gray), CTNNB1 KO (dark purple) and CTNNB1 Udel (dark blue) clones at the iPSC stage. The unstrained samples are shown in lighter colors. D. Quantification of the experiment from (C). Surface E-cadherin abundance was measured by MFI. Data are shown as mean ± SD from N = 3 independent experiments of N = 3 clonal lines, each. Welch’s t -test. ****, P = 2.5 x 10 -7 . E. Quantification of nuclear β-catenin relative to total β-catenin at 24h of DE differentiation from immunoblot analysis, shown in . Data are shown as mean ± SD from N = 2 independent experiments. Welch’s t- test. ns, not significant. F. Representative immunofluorescence images showing β-catenin distribution at 4h of DE differentiation. DAPI was used to stain the nucleus. Scale bar, 10 μm. G. Quantification of nuclear β-catenin from the experiment shown in (F). For each experiment, 8–10 random fields (>80 cells per experiment) were analyzed. Each dot represents the mean intensity from one experiment. Data are shown as mean ± SD from N = 3 independent experiments. Welch’s t -test. ns, not significant. H. Sanger sequencing of edited alleles of zebrafish ctnnb1 KO embryos. The <t>guide</t> <t>RNA</t> is underlined, and the cut site is indicated by the vertical dashed line. I. Representative gel image of genotyping PCR to validate zebrafish ctnnb1 3′UTR deletions using deletion-flanking primers. J. Schematic of the zebrafish ctnnb1 3′UTR deletion region. The predicted post-editing sequence is shown. Red arrowheads indicate the junction site after scarless repair. In mosaic embryos, edited PCR products were excised and analyzed by Amplicon sequencing. Sequences were aligned and composition is shown. Sequences matching the prediction are underlined; insertions are highlighted in red. K. As in , but additional zebrafish images are shown.
    Guide Rna Pairs, supplied by Synthego Inc, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/guide rna pairs/product/Synthego Inc
    Average 86 stars, based on 1 article reviews
    guide rna pairs - by Bioz Stars, 2026-06
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    Images

    1) Product Images from "Intermolecular 3′UTR-3′UTR interactions drive Wnt gene activation through heteromeric protein assembly"

    Article Title: Intermolecular 3′UTR-3′UTR interactions drive Wnt gene activation through heteromeric protein assembly

    Journal: bioRxiv

    doi: 10.64898/2026.05.05.723075

    A. Schematic of β-catenin interaction with E-cadherin at the plasma membrane. B. Immunoblot analysis of total E-cadherin and β-catenin protein abundance in iPSCs. N = 3 clonal lines each were used, and Tubulin serves as loading control. C. Representative flow plots showing surface E-cadherin in control (dark gray), CTNNB1 KO (dark purple) and CTNNB1 Udel (dark blue) clones at the iPSC stage. The unstrained samples are shown in lighter colors. D. Quantification of the experiment from (C). Surface E-cadherin abundance was measured by MFI. Data are shown as mean ± SD from N = 3 independent experiments of N = 3 clonal lines, each. Welch’s t -test. ****, P = 2.5 x 10 -7 . E. Quantification of nuclear β-catenin relative to total β-catenin at 24h of DE differentiation from immunoblot analysis, shown in . Data are shown as mean ± SD from N = 2 independent experiments. Welch’s t- test. ns, not significant. F. Representative immunofluorescence images showing β-catenin distribution at 4h of DE differentiation. DAPI was used to stain the nucleus. Scale bar, 10 μm. G. Quantification of nuclear β-catenin from the experiment shown in (F). For each experiment, 8–10 random fields (>80 cells per experiment) were analyzed. Each dot represents the mean intensity from one experiment. Data are shown as mean ± SD from N = 3 independent experiments. Welch’s t -test. ns, not significant. H. Sanger sequencing of edited alleles of zebrafish ctnnb1 KO embryos. The guide RNA is underlined, and the cut site is indicated by the vertical dashed line. I. Representative gel image of genotyping PCR to validate zebrafish ctnnb1 3′UTR deletions using deletion-flanking primers. J. Schematic of the zebrafish ctnnb1 3′UTR deletion region. The predicted post-editing sequence is shown. Red arrowheads indicate the junction site after scarless repair. In mosaic embryos, edited PCR products were excised and analyzed by Amplicon sequencing. Sequences were aligned and composition is shown. Sequences matching the prediction are underlined; insertions are highlighted in red. K. As in , but additional zebrafish images are shown.
    Figure Legend Snippet: A. Schematic of β-catenin interaction with E-cadherin at the plasma membrane. B. Immunoblot analysis of total E-cadherin and β-catenin protein abundance in iPSCs. N = 3 clonal lines each were used, and Tubulin serves as loading control. C. Representative flow plots showing surface E-cadherin in control (dark gray), CTNNB1 KO (dark purple) and CTNNB1 Udel (dark blue) clones at the iPSC stage. The unstrained samples are shown in lighter colors. D. Quantification of the experiment from (C). Surface E-cadherin abundance was measured by MFI. Data are shown as mean ± SD from N = 3 independent experiments of N = 3 clonal lines, each. Welch’s t -test. ****, P = 2.5 x 10 -7 . E. Quantification of nuclear β-catenin relative to total β-catenin at 24h of DE differentiation from immunoblot analysis, shown in . Data are shown as mean ± SD from N = 2 independent experiments. Welch’s t- test. ns, not significant. F. Representative immunofluorescence images showing β-catenin distribution at 4h of DE differentiation. DAPI was used to stain the nucleus. Scale bar, 10 μm. G. Quantification of nuclear β-catenin from the experiment shown in (F). For each experiment, 8–10 random fields (>80 cells per experiment) were analyzed. Each dot represents the mean intensity from one experiment. Data are shown as mean ± SD from N = 3 independent experiments. Welch’s t -test. ns, not significant. H. Sanger sequencing of edited alleles of zebrafish ctnnb1 KO embryos. The guide RNA is underlined, and the cut site is indicated by the vertical dashed line. I. Representative gel image of genotyping PCR to validate zebrafish ctnnb1 3′UTR deletions using deletion-flanking primers. J. Schematic of the zebrafish ctnnb1 3′UTR deletion region. The predicted post-editing sequence is shown. Red arrowheads indicate the junction site after scarless repair. In mosaic embryos, edited PCR products were excised and analyzed by Amplicon sequencing. Sequences were aligned and composition is shown. Sequences matching the prediction are underlined; insertions are highlighted in red. K. As in , but additional zebrafish images are shown.

    Techniques Used: Clinical Proteomics, Membrane, Western Blot, Quantitative Proteomics, Control, Clone Assay, Immunofluorescence, Staining, Sequencing, Amplification

    A. Schematic of β-catenin-mediated activation of the Wnt transcriptional program. B. LEF1 mRNA expression at the indicated time points, normalized to GAPDH . Shown is mean ± SD rom N = 3 independent experiments. Welch’s t -test; *, P < 0.05; **, P < 0.01; ****, P < 0.0001. C. As in (B), but AXIN2 mRNA expression is shown. D. Immunoblot showing total β-catenin in Ctrl and Udel cells at the indicated time points. N = 2 clonal lines were examined. Tubulin serves as loading control. E. Immunoblot showing active and total β-catenin in Ctrl and Udel cells at DE 24h. N = 3 clonal lines were examined. Tubulin serves as loading control. F. Immunoblot showing nuclear and cytoplasmic β-catenin at DE 24h loaded at 1:1 ratio. Tubulin serves as loading control for cytoplasmic fraction and H3 serves as loading control for nuclear fraction. Quantification, see . G. Scatter plot showing log2FC in Udel versus Ctrl (x-axis) and log2FC in KO versus Ctrl (y-axis) at DE 24h of Wnt-responsive genes . Genes with significant changes (log2|FC| > 0.58 and FDR < 0.05) in both Udel and KO are shown in dark blue ( N = 231), whereas genes with significant change in KO only are shown in light blue ( N = 1933). Dashed lines indicate a FC of 1.5. Selected genes are indicated. Pearson’s correlation coefficient is shown. H. Gene ontology analysis of genes colored in (G). Bonferroni-corrected P values are shown. I. Shown is mean log2FC of Wnt-responsive genes with significant gene expression changes between DE 24h and stem cell state in Ctrl clones, stratified by the magnitude of induction or repression. The number of genes in the eight bins are 15, 21, 87, 290, 215, 75, 35, and 57. T-test for independent samples; ****, P < 2 x 10 -9 . J. For the genes from (I), mean log2FC in Udel versus Ctrl cells at DE 24h is shown. T-test for independent samples; *, P = 0.046; ***, P = 0.008. K. Schematic of 3′UTR loss-of-function approach of the zebrafish ctnnb1 gene. A genomic region of 776 bp is deleted using CRISPR-Cas9 and a pair of guide RNAs in fertilized eggs. At the mRNA level, this deletion results in partial deletion of the zebrafish ctnnb1 3′UTR. Embryonic defects are scored 72h after fertilization. Top panel, conserved nt between the human CTNNB1 and the zebrafish ctnnb1 3′UTR. Each line denotes an identical nt. L. Representative images showing a normal zebrafish embryo, injected with a non-targeting guide RNA (Ctrl), mild and severe abnormalities observed after injection of a pair of guide RNAs that generate a ctnnb1 3′UTR deletion (Udel) and severe abnormalities after injection of a guide RNA targeting the ctnnb1 coding sequence to generate a gene KO. Scale bar, 500 μm. M. Phenotype classification at 72h post-injection from experiment shown in (L). The total number of fish examined in each category is given. Shown is the mean fraction ± SD of the obtained phenotypes from three clutches obtained in two independent experiments. T-test for independent samples was performed; mild phenotype, uninjected (uninj) vs Udel, **, P =0.008; Uninj vs KO, ns; severe phenotype, uninj vs Udel, *, P = 0.046; uninj vs KO, ****, P = 4 x 10 -6 . N. Immunoblot showing total β-catenin obtained from zebrafish embryos at 72h post-injection. Four embryos were pooled for each sample. Actin was used as loading control. The numbers indicate relative protein abundance normalized to Actin in each sample. O. mRNA expression of lef1 and axin2 in zebrafish embryos 72 h post-injection, normalized to eef1 . Shown is mean ± SD of N = 3 mRNA preparations that each contained four different embryos. Welch’s t -test; *, P < 0.05; **, P < 0.01.
    Figure Legend Snippet: A. Schematic of β-catenin-mediated activation of the Wnt transcriptional program. B. LEF1 mRNA expression at the indicated time points, normalized to GAPDH . Shown is mean ± SD rom N = 3 independent experiments. Welch’s t -test; *, P < 0.05; **, P < 0.01; ****, P < 0.0001. C. As in (B), but AXIN2 mRNA expression is shown. D. Immunoblot showing total β-catenin in Ctrl and Udel cells at the indicated time points. N = 2 clonal lines were examined. Tubulin serves as loading control. E. Immunoblot showing active and total β-catenin in Ctrl and Udel cells at DE 24h. N = 3 clonal lines were examined. Tubulin serves as loading control. F. Immunoblot showing nuclear and cytoplasmic β-catenin at DE 24h loaded at 1:1 ratio. Tubulin serves as loading control for cytoplasmic fraction and H3 serves as loading control for nuclear fraction. Quantification, see . G. Scatter plot showing log2FC in Udel versus Ctrl (x-axis) and log2FC in KO versus Ctrl (y-axis) at DE 24h of Wnt-responsive genes . Genes with significant changes (log2|FC| > 0.58 and FDR < 0.05) in both Udel and KO are shown in dark blue ( N = 231), whereas genes with significant change in KO only are shown in light blue ( N = 1933). Dashed lines indicate a FC of 1.5. Selected genes are indicated. Pearson’s correlation coefficient is shown. H. Gene ontology analysis of genes colored in (G). Bonferroni-corrected P values are shown. I. Shown is mean log2FC of Wnt-responsive genes with significant gene expression changes between DE 24h and stem cell state in Ctrl clones, stratified by the magnitude of induction or repression. The number of genes in the eight bins are 15, 21, 87, 290, 215, 75, 35, and 57. T-test for independent samples; ****, P < 2 x 10 -9 . J. For the genes from (I), mean log2FC in Udel versus Ctrl cells at DE 24h is shown. T-test for independent samples; *, P = 0.046; ***, P = 0.008. K. Schematic of 3′UTR loss-of-function approach of the zebrafish ctnnb1 gene. A genomic region of 776 bp is deleted using CRISPR-Cas9 and a pair of guide RNAs in fertilized eggs. At the mRNA level, this deletion results in partial deletion of the zebrafish ctnnb1 3′UTR. Embryonic defects are scored 72h after fertilization. Top panel, conserved nt between the human CTNNB1 and the zebrafish ctnnb1 3′UTR. Each line denotes an identical nt. L. Representative images showing a normal zebrafish embryo, injected with a non-targeting guide RNA (Ctrl), mild and severe abnormalities observed after injection of a pair of guide RNAs that generate a ctnnb1 3′UTR deletion (Udel) and severe abnormalities after injection of a guide RNA targeting the ctnnb1 coding sequence to generate a gene KO. Scale bar, 500 μm. M. Phenotype classification at 72h post-injection from experiment shown in (L). The total number of fish examined in each category is given. Shown is the mean fraction ± SD of the obtained phenotypes from three clutches obtained in two independent experiments. T-test for independent samples was performed; mild phenotype, uninjected (uninj) vs Udel, **, P =0.008; Uninj vs KO, ns; severe phenotype, uninj vs Udel, *, P = 0.046; uninj vs KO, ****, P = 4 x 10 -6 . N. Immunoblot showing total β-catenin obtained from zebrafish embryos at 72h post-injection. Four embryos were pooled for each sample. Actin was used as loading control. The numbers indicate relative protein abundance normalized to Actin in each sample. O. mRNA expression of lef1 and axin2 in zebrafish embryos 72 h post-injection, normalized to eef1 . Shown is mean ± SD of N = 3 mRNA preparations that each contained four different embryos. Welch’s t -test; *, P < 0.05; **, P < 0.01.

    Techniques Used: Activation Assay, Expressing, Western Blot, Control, Gene Expression, Clone Assay, CRISPR, Injection, Sequencing, Quantitative Proteomics



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    Danaher Inc alt r synthetic paired guide rnas
    lncRNA dropout CRISPR-Cas9 screen in MM cell lines. (A) Schematic representation of the CRISPR screening pipeline. (B) Spearman's correlation between <t>pgRNA</t> read count profiles (from DNA collected 30 days after transduction and selection of the library) across screen replicates = 0.85 and 0.88, respectively, for AMO-1 and ABZB, with color bars on top/left indicating cluster membership obtained via hierarchical clustering (complete distance method). (C) Representation of pgRNA abundance log fold changes (logFCs) in DNA collected 30 days after library transduction and selection vs plasmidic amounts for 3 groups of pgRNAs: nontargeting (negative controls [median logFC = 0.27 and 0.35, respectively, for AMO-1 and ABZB]), targeting ribosomal protein genes (control essential genes, median logFC = −0.68 and −0.43, with a logFC ≤−0.5, corresponding to a MAGeCK FDR ≤20%), and lncRNAs, across the 2 screens. Each point represents 1 of the 12 472 pgRNAs in the library with coordinates on the y-axis indicating the median logFC across screen replicates. (D) Gene-wise MAGeCK robust rank aggregation (RRA) scores for significant dependencies identified in the 2 screens at an FDR ≤20%. Top essential control genes, dependencies that are private to each cell line and shared across them (as per the color scheme) are highligted. (E) Number of significantly essential lncRNAs (at an FDR ≤20%) in the 2 screened cell lines and their overlap. MOI, multiplicity of infection.
    Alt R Synthetic Paired Guide Rnas, supplied by Danaher Inc, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/alt r synthetic paired guide rnas/product/Danaher Inc
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    single-guide (sg)rna pair trueguide synthetic guide rna  (Thermo Fisher)
    90
    Thermo Fisher single-guide (sg)rna pair trueguide synthetic guide rna
    lncRNA dropout CRISPR-Cas9 screen in MM cell lines. (A) Schematic representation of the CRISPR screening pipeline. (B) Spearman's correlation between <t>pgRNA</t> read count profiles (from DNA collected 30 days after transduction and selection of the library) across screen replicates = 0.85 and 0.88, respectively, for AMO-1 and ABZB, with color bars on top/left indicating cluster membership obtained via hierarchical clustering (complete distance method). (C) Representation of pgRNA abundance log fold changes (logFCs) in DNA collected 30 days after library transduction and selection vs plasmidic amounts for 3 groups of pgRNAs: nontargeting (negative controls [median logFC = 0.27 and 0.35, respectively, for AMO-1 and ABZB]), targeting ribosomal protein genes (control essential genes, median logFC = −0.68 and −0.43, with a logFC ≤−0.5, corresponding to a MAGeCK FDR ≤20%), and lncRNAs, across the 2 screens. Each point represents 1 of the 12 472 pgRNAs in the library with coordinates on the y-axis indicating the median logFC across screen replicates. (D) Gene-wise MAGeCK robust rank aggregation (RRA) scores for significant dependencies identified in the 2 screens at an FDR ≤20%. Top essential control genes, dependencies that are private to each cell line and shared across them (as per the color scheme) are highligted. (E) Number of significantly essential lncRNAs (at an FDR ≤20%) in the 2 screened cell lines and their overlap. MOI, multiplicity of infection.
    Single Guide (Sg)rna Pair Trueguide Synthetic Guide Rna, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/single-guide (sg)rna pair trueguide synthetic guide rna/product/Thermo Fisher
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    plasmids encoding paired guide rnas targeting the 10th exon of the casp8ap2 gene  (Transomic Technologies Inc)
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    Transomic Technologies Inc plasmids encoding paired guide rnas targeting the 10th exon of the casp8ap2 gene
    Knockout of the <t>CASP8AP2</t> gene. (a) Agarose gel electrophoresis of polymerase chain reaction (PCR) amplified CASP8AP2 locus in the parental and the 892–7 cell lines, and EMBOSS WATER sequence alignment of both alleles with the parental sequence. Guide RNA (gRNA) target sequence is highlighted. GAPDH-normalized qPCR analysis of CASP8AP2 messenger RNA (mRNA) abundance in parental and CASP8AP2 knockout clone, 892–7, using primers targeting the gRNA locus for amplification. (b) Caspase 8 activity in cell lysates of control (uninduced) and apoptosis-induced (induced) 892–7 and parental cells (**p < .001)
    Plasmids Encoding Paired Guide Rnas Targeting The 10th Exon Of The Casp8ap2 Gene, supplied by Transomic Technologies Inc, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/plasmids encoding paired guide rnas targeting the 10th exon of the casp8ap2 gene/product/Transomic Technologies Inc
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    Image Search Results


    A. Schematic of β-catenin interaction with E-cadherin at the plasma membrane. B. Immunoblot analysis of total E-cadherin and β-catenin protein abundance in iPSCs. N = 3 clonal lines each were used, and Tubulin serves as loading control. C. Representative flow plots showing surface E-cadherin in control (dark gray), CTNNB1 KO (dark purple) and CTNNB1 Udel (dark blue) clones at the iPSC stage. The unstrained samples are shown in lighter colors. D. Quantification of the experiment from (C). Surface E-cadherin abundance was measured by MFI. Data are shown as mean ± SD from N = 3 independent experiments of N = 3 clonal lines, each. Welch’s t -test. ****, P = 2.5 x 10 -7 . E. Quantification of nuclear β-catenin relative to total β-catenin at 24h of DE differentiation from immunoblot analysis, shown in . Data are shown as mean ± SD from N = 2 independent experiments. Welch’s t- test. ns, not significant. F. Representative immunofluorescence images showing β-catenin distribution at 4h of DE differentiation. DAPI was used to stain the nucleus. Scale bar, 10 μm. G. Quantification of nuclear β-catenin from the experiment shown in (F). For each experiment, 8–10 random fields (>80 cells per experiment) were analyzed. Each dot represents the mean intensity from one experiment. Data are shown as mean ± SD from N = 3 independent experiments. Welch’s t -test. ns, not significant. H. Sanger sequencing of edited alleles of zebrafish ctnnb1 KO embryos. The guide RNA is underlined, and the cut site is indicated by the vertical dashed line. I. Representative gel image of genotyping PCR to validate zebrafish ctnnb1 3′UTR deletions using deletion-flanking primers. J. Schematic of the zebrafish ctnnb1 3′UTR deletion region. The predicted post-editing sequence is shown. Red arrowheads indicate the junction site after scarless repair. In mosaic embryos, edited PCR products were excised and analyzed by Amplicon sequencing. Sequences were aligned and composition is shown. Sequences matching the prediction are underlined; insertions are highlighted in red. K. As in , but additional zebrafish images are shown.

    Journal: bioRxiv

    Article Title: Intermolecular 3′UTR-3′UTR interactions drive Wnt gene activation through heteromeric protein assembly

    doi: 10.64898/2026.05.05.723075

    Figure Lengend Snippet: A. Schematic of β-catenin interaction with E-cadherin at the plasma membrane. B. Immunoblot analysis of total E-cadherin and β-catenin protein abundance in iPSCs. N = 3 clonal lines each were used, and Tubulin serves as loading control. C. Representative flow plots showing surface E-cadherin in control (dark gray), CTNNB1 KO (dark purple) and CTNNB1 Udel (dark blue) clones at the iPSC stage. The unstrained samples are shown in lighter colors. D. Quantification of the experiment from (C). Surface E-cadherin abundance was measured by MFI. Data are shown as mean ± SD from N = 3 independent experiments of N = 3 clonal lines, each. Welch’s t -test. ****, P = 2.5 x 10 -7 . E. Quantification of nuclear β-catenin relative to total β-catenin at 24h of DE differentiation from immunoblot analysis, shown in . Data are shown as mean ± SD from N = 2 independent experiments. Welch’s t- test. ns, not significant. F. Representative immunofluorescence images showing β-catenin distribution at 4h of DE differentiation. DAPI was used to stain the nucleus. Scale bar, 10 μm. G. Quantification of nuclear β-catenin from the experiment shown in (F). For each experiment, 8–10 random fields (>80 cells per experiment) were analyzed. Each dot represents the mean intensity from one experiment. Data are shown as mean ± SD from N = 3 independent experiments. Welch’s t -test. ns, not significant. H. Sanger sequencing of edited alleles of zebrafish ctnnb1 KO embryos. The guide RNA is underlined, and the cut site is indicated by the vertical dashed line. I. Representative gel image of genotyping PCR to validate zebrafish ctnnb1 3′UTR deletions using deletion-flanking primers. J. Schematic of the zebrafish ctnnb1 3′UTR deletion region. The predicted post-editing sequence is shown. Red arrowheads indicate the junction site after scarless repair. In mosaic embryos, edited PCR products were excised and analyzed by Amplicon sequencing. Sequences were aligned and composition is shown. Sequences matching the prediction are underlined; insertions are highlighted in red. K. As in , but additional zebrafish images are shown.

    Article Snippet: The guide RNA pairs with the highest editing efficiency were purchased from Synthego (Table S6).

    Techniques: Clinical Proteomics, Membrane, Western Blot, Quantitative Proteomics, Control, Clone Assay, Immunofluorescence, Staining, Sequencing, Amplification

    A. Schematic of β-catenin-mediated activation of the Wnt transcriptional program. B. LEF1 mRNA expression at the indicated time points, normalized to GAPDH . Shown is mean ± SD rom N = 3 independent experiments. Welch’s t -test; *, P < 0.05; **, P < 0.01; ****, P < 0.0001. C. As in (B), but AXIN2 mRNA expression is shown. D. Immunoblot showing total β-catenin in Ctrl and Udel cells at the indicated time points. N = 2 clonal lines were examined. Tubulin serves as loading control. E. Immunoblot showing active and total β-catenin in Ctrl and Udel cells at DE 24h. N = 3 clonal lines were examined. Tubulin serves as loading control. F. Immunoblot showing nuclear and cytoplasmic β-catenin at DE 24h loaded at 1:1 ratio. Tubulin serves as loading control for cytoplasmic fraction and H3 serves as loading control for nuclear fraction. Quantification, see . G. Scatter plot showing log2FC in Udel versus Ctrl (x-axis) and log2FC in KO versus Ctrl (y-axis) at DE 24h of Wnt-responsive genes . Genes with significant changes (log2|FC| > 0.58 and FDR < 0.05) in both Udel and KO are shown in dark blue ( N = 231), whereas genes with significant change in KO only are shown in light blue ( N = 1933). Dashed lines indicate a FC of 1.5. Selected genes are indicated. Pearson’s correlation coefficient is shown. H. Gene ontology analysis of genes colored in (G). Bonferroni-corrected P values are shown. I. Shown is mean log2FC of Wnt-responsive genes with significant gene expression changes between DE 24h and stem cell state in Ctrl clones, stratified by the magnitude of induction or repression. The number of genes in the eight bins are 15, 21, 87, 290, 215, 75, 35, and 57. T-test for independent samples; ****, P < 2 x 10 -9 . J. For the genes from (I), mean log2FC in Udel versus Ctrl cells at DE 24h is shown. T-test for independent samples; *, P = 0.046; ***, P = 0.008. K. Schematic of 3′UTR loss-of-function approach of the zebrafish ctnnb1 gene. A genomic region of 776 bp is deleted using CRISPR-Cas9 and a pair of guide RNAs in fertilized eggs. At the mRNA level, this deletion results in partial deletion of the zebrafish ctnnb1 3′UTR. Embryonic defects are scored 72h after fertilization. Top panel, conserved nt between the human CTNNB1 and the zebrafish ctnnb1 3′UTR. Each line denotes an identical nt. L. Representative images showing a normal zebrafish embryo, injected with a non-targeting guide RNA (Ctrl), mild and severe abnormalities observed after injection of a pair of guide RNAs that generate a ctnnb1 3′UTR deletion (Udel) and severe abnormalities after injection of a guide RNA targeting the ctnnb1 coding sequence to generate a gene KO. Scale bar, 500 μm. M. Phenotype classification at 72h post-injection from experiment shown in (L). The total number of fish examined in each category is given. Shown is the mean fraction ± SD of the obtained phenotypes from three clutches obtained in two independent experiments. T-test for independent samples was performed; mild phenotype, uninjected (uninj) vs Udel, **, P =0.008; Uninj vs KO, ns; severe phenotype, uninj vs Udel, *, P = 0.046; uninj vs KO, ****, P = 4 x 10 -6 . N. Immunoblot showing total β-catenin obtained from zebrafish embryos at 72h post-injection. Four embryos were pooled for each sample. Actin was used as loading control. The numbers indicate relative protein abundance normalized to Actin in each sample. O. mRNA expression of lef1 and axin2 in zebrafish embryos 72 h post-injection, normalized to eef1 . Shown is mean ± SD of N = 3 mRNA preparations that each contained four different embryos. Welch’s t -test; *, P < 0.05; **, P < 0.01.

    Journal: bioRxiv

    Article Title: Intermolecular 3′UTR-3′UTR interactions drive Wnt gene activation through heteromeric protein assembly

    doi: 10.64898/2026.05.05.723075

    Figure Lengend Snippet: A. Schematic of β-catenin-mediated activation of the Wnt transcriptional program. B. LEF1 mRNA expression at the indicated time points, normalized to GAPDH . Shown is mean ± SD rom N = 3 independent experiments. Welch’s t -test; *, P < 0.05; **, P < 0.01; ****, P < 0.0001. C. As in (B), but AXIN2 mRNA expression is shown. D. Immunoblot showing total β-catenin in Ctrl and Udel cells at the indicated time points. N = 2 clonal lines were examined. Tubulin serves as loading control. E. Immunoblot showing active and total β-catenin in Ctrl and Udel cells at DE 24h. N = 3 clonal lines were examined. Tubulin serves as loading control. F. Immunoblot showing nuclear and cytoplasmic β-catenin at DE 24h loaded at 1:1 ratio. Tubulin serves as loading control for cytoplasmic fraction and H3 serves as loading control for nuclear fraction. Quantification, see . G. Scatter plot showing log2FC in Udel versus Ctrl (x-axis) and log2FC in KO versus Ctrl (y-axis) at DE 24h of Wnt-responsive genes . Genes with significant changes (log2|FC| > 0.58 and FDR < 0.05) in both Udel and KO are shown in dark blue ( N = 231), whereas genes with significant change in KO only are shown in light blue ( N = 1933). Dashed lines indicate a FC of 1.5. Selected genes are indicated. Pearson’s correlation coefficient is shown. H. Gene ontology analysis of genes colored in (G). Bonferroni-corrected P values are shown. I. Shown is mean log2FC of Wnt-responsive genes with significant gene expression changes between DE 24h and stem cell state in Ctrl clones, stratified by the magnitude of induction or repression. The number of genes in the eight bins are 15, 21, 87, 290, 215, 75, 35, and 57. T-test for independent samples; ****, P < 2 x 10 -9 . J. For the genes from (I), mean log2FC in Udel versus Ctrl cells at DE 24h is shown. T-test for independent samples; *, P = 0.046; ***, P = 0.008. K. Schematic of 3′UTR loss-of-function approach of the zebrafish ctnnb1 gene. A genomic region of 776 bp is deleted using CRISPR-Cas9 and a pair of guide RNAs in fertilized eggs. At the mRNA level, this deletion results in partial deletion of the zebrafish ctnnb1 3′UTR. Embryonic defects are scored 72h after fertilization. Top panel, conserved nt between the human CTNNB1 and the zebrafish ctnnb1 3′UTR. Each line denotes an identical nt. L. Representative images showing a normal zebrafish embryo, injected with a non-targeting guide RNA (Ctrl), mild and severe abnormalities observed after injection of a pair of guide RNAs that generate a ctnnb1 3′UTR deletion (Udel) and severe abnormalities after injection of a guide RNA targeting the ctnnb1 coding sequence to generate a gene KO. Scale bar, 500 μm. M. Phenotype classification at 72h post-injection from experiment shown in (L). The total number of fish examined in each category is given. Shown is the mean fraction ± SD of the obtained phenotypes from three clutches obtained in two independent experiments. T-test for independent samples was performed; mild phenotype, uninjected (uninj) vs Udel, **, P =0.008; Uninj vs KO, ns; severe phenotype, uninj vs Udel, *, P = 0.046; uninj vs KO, ****, P = 4 x 10 -6 . N. Immunoblot showing total β-catenin obtained from zebrafish embryos at 72h post-injection. Four embryos were pooled for each sample. Actin was used as loading control. The numbers indicate relative protein abundance normalized to Actin in each sample. O. mRNA expression of lef1 and axin2 in zebrafish embryos 72 h post-injection, normalized to eef1 . Shown is mean ± SD of N = 3 mRNA preparations that each contained four different embryos. Welch’s t -test; *, P < 0.05; **, P < 0.01.

    Article Snippet: The guide RNA pairs with the highest editing efficiency were purchased from Synthego (Table S6).

    Techniques: Activation Assay, Expressing, Western Blot, Control, Gene Expression, Clone Assay, CRISPR, Injection, Sequencing, Quantitative Proteomics

    lncRNA dropout CRISPR-Cas9 screen in MM cell lines. (A) Schematic representation of the CRISPR screening pipeline. (B) Spearman's correlation between pgRNA read count profiles (from DNA collected 30 days after transduction and selection of the library) across screen replicates = 0.85 and 0.88, respectively, for AMO-1 and ABZB, with color bars on top/left indicating cluster membership obtained via hierarchical clustering (complete distance method). (C) Representation of pgRNA abundance log fold changes (logFCs) in DNA collected 30 days after library transduction and selection vs plasmidic amounts for 3 groups of pgRNAs: nontargeting (negative controls [median logFC = 0.27 and 0.35, respectively, for AMO-1 and ABZB]), targeting ribosomal protein genes (control essential genes, median logFC = −0.68 and −0.43, with a logFC ≤−0.5, corresponding to a MAGeCK FDR ≤20%), and lncRNAs, across the 2 screens. Each point represents 1 of the 12 472 pgRNAs in the library with coordinates on the y-axis indicating the median logFC across screen replicates. (D) Gene-wise MAGeCK robust rank aggregation (RRA) scores for significant dependencies identified in the 2 screens at an FDR ≤20%. Top essential control genes, dependencies that are private to each cell line and shared across them (as per the color scheme) are highligted. (E) Number of significantly essential lncRNAs (at an FDR ≤20%) in the 2 screened cell lines and their overlap. MOI, multiplicity of infection.

    Journal: Blood

    Article Title: An unbiased lncRNA dropout CRISPR-Cas9 screen reveals RP11-350G8.5 as a novel therapeutic target for multiple myeloma ∗

    doi: 10.1182/blood.2023021991

    Figure Lengend Snippet: lncRNA dropout CRISPR-Cas9 screen in MM cell lines. (A) Schematic representation of the CRISPR screening pipeline. (B) Spearman's correlation between pgRNA read count profiles (from DNA collected 30 days after transduction and selection of the library) across screen replicates = 0.85 and 0.88, respectively, for AMO-1 and ABZB, with color bars on top/left indicating cluster membership obtained via hierarchical clustering (complete distance method). (C) Representation of pgRNA abundance log fold changes (logFCs) in DNA collected 30 days after library transduction and selection vs plasmidic amounts for 3 groups of pgRNAs: nontargeting (negative controls [median logFC = 0.27 and 0.35, respectively, for AMO-1 and ABZB]), targeting ribosomal protein genes (control essential genes, median logFC = −0.68 and −0.43, with a logFC ≤−0.5, corresponding to a MAGeCK FDR ≤20%), and lncRNAs, across the 2 screens. Each point represents 1 of the 12 472 pgRNAs in the library with coordinates on the y-axis indicating the median logFC across screen replicates. (D) Gene-wise MAGeCK robust rank aggregation (RRA) scores for significant dependencies identified in the 2 screens at an FDR ≤20%. Top essential control genes, dependencies that are private to each cell line and shared across them (as per the color scheme) are highligted. (E) Number of significantly essential lncRNAs (at an FDR ≤20%) in the 2 screened cell lines and their overlap. MOI, multiplicity of infection.

    Article Snippet: The human paired-guide RNA (pgRNA) library pool ( ) (Addgene number 89640) was used to perform the CRISPR-Cas9 screens (supplemental Materials and methods; , and ).

    Techniques: CRISPR, Transduction, Selection, Control, Infection

    Functional validation of prioritized oncogenic lncRNA candidates. (A) RP11-350G8.5 and LINC00467 basal expression levels via quantitative real time PCR (qRT-PCR) in MM cell lines and peripheral blood mononuclear cells (PBMCs) from healthy donors (values are normalized to the expression of GAPDH). (B) Representative image of genomic PCR products before and after KO of LINC00467 and RP11-350G8.5 in AMO-1 cells, visualized on 1.5% agarose gels. On the right: Sanger sequence of the amplicons encompassing the CRISPR-targeted region. Blue rectangles highlight pgRNA binding sites, whereas colored lines refer to the schematic picture of the KO reported above the gel picture (on the left). (C) Representative image of flow cytometric monitoring of AMO-1 and ABZB cells transduced with a SCRAMBLE-GFP-CRISPR vector (dark gray) or LINC00467/KO-GFP-CRISPR vector (light blue) or RP11-350G8.5/KO- GFP-CRISPR vector (red). Light-gray curves represent the percentage of viable cells at day 0 (48 hours after lentiviral transduction) with overlapping colored curves at day 20. (D) Representative images of colony assay of AMO-1 and ABZB GFP-sorted cells, 15 days after plating, were generated using EVOS XL-Core microscope (Invitrogen by Thermo Fisher) (magnification ×10). (E) Number of colonies in 3 independent wells. (F) Dose-response curves 24 hours after treatment with bortezomib (1-10 nM). Percentage of viable cells ± standard deviation are normalized with respect to DMSO-treated cells (vehicle) for each experimental condition. Statistical differences were assessed across all plots via Student t test; ∗ P < .05, ∗∗ P < .01, and ∗∗∗ P < .001.

    Journal: Blood

    Article Title: An unbiased lncRNA dropout CRISPR-Cas9 screen reveals RP11-350G8.5 as a novel therapeutic target for multiple myeloma ∗

    doi: 10.1182/blood.2023021991

    Figure Lengend Snippet: Functional validation of prioritized oncogenic lncRNA candidates. (A) RP11-350G8.5 and LINC00467 basal expression levels via quantitative real time PCR (qRT-PCR) in MM cell lines and peripheral blood mononuclear cells (PBMCs) from healthy donors (values are normalized to the expression of GAPDH). (B) Representative image of genomic PCR products before and after KO of LINC00467 and RP11-350G8.5 in AMO-1 cells, visualized on 1.5% agarose gels. On the right: Sanger sequence of the amplicons encompassing the CRISPR-targeted region. Blue rectangles highlight pgRNA binding sites, whereas colored lines refer to the schematic picture of the KO reported above the gel picture (on the left). (C) Representative image of flow cytometric monitoring of AMO-1 and ABZB cells transduced with a SCRAMBLE-GFP-CRISPR vector (dark gray) or LINC00467/KO-GFP-CRISPR vector (light blue) or RP11-350G8.5/KO- GFP-CRISPR vector (red). Light-gray curves represent the percentage of viable cells at day 0 (48 hours after lentiviral transduction) with overlapping colored curves at day 20. (D) Representative images of colony assay of AMO-1 and ABZB GFP-sorted cells, 15 days after plating, were generated using EVOS XL-Core microscope (Invitrogen by Thermo Fisher) (magnification ×10). (E) Number of colonies in 3 independent wells. (F) Dose-response curves 24 hours after treatment with bortezomib (1-10 nM). Percentage of viable cells ± standard deviation are normalized with respect to DMSO-treated cells (vehicle) for each experimental condition. Statistical differences were assessed across all plots via Student t test; ∗ P < .05, ∗∗ P < .01, and ∗∗∗ P < .001.

    Article Snippet: The human paired-guide RNA (pgRNA) library pool ( ) (Addgene number 89640) was used to perform the CRISPR-Cas9 screens (supplemental Materials and methods; , and ).

    Techniques: Functional Assay, Biomarker Discovery, Expressing, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Sequencing, CRISPR, Binding Assay, Transduction, Plasmid Preparation, Colony Assay, Generated, Microscopy, Standard Deviation

    RP11-350G8.5 putative oncogenic role: in vitro validation and preliminary data from in vivo models. (A) Flow cytometric monitoring of GFP expression in ABZB cells transduced with a SCRAMBLE-GFP-CRISPR negative control vector, an RPL8 /KO-GFP-CRISPR positive control vector (selected from Project Score [37]), and 2 GFP-CRISPR constructs encoding for 2 pgRNAs targeting RP11-350G8.5. Gray curves represent the percentage of viable cells at day 0 (48 hours after lentiviral transduction), while colored curves represent the percentage of viable cells at day 20. Bars on the right represent the fold change in percentage of GFP-expressing cells 20 days after target depletion against day 0. (B) Evaluation of IL-6R RNA expression level through quantitative real time PCR (qRT-PCR) on ABZB after transduction with SCRAMBLE vector or KO of RP11-350G8.5 with pgRNA#1 or pgRNA#2 or with a vector overexpressing RP11-350G8.5 (UP). (Data are normalized to the expression of GAPDH.) Statistics were obtained using multiple t -tests, resulting in no significant (ns) differences, as per the reported P values. (C) Flow cytometric monitoring of GFP in JJN.3 and NCI-H929 MM transduced cells, and percentage of GFP-positive cells is reported by overlapping curves referred to day 20 (colored curves) against day 0 (light gray curves). (D) Validation of RP11-350G8.5 KO in nontumoral cells, performed as described for A and C. (E) Representative images of RNA-FISH analysis. Nuclei are counterstained with DAPI (blue signal), whereas C3-fluorescein–conjugated GAPDH (green signal) has been used as cytoplasmic marker. Customly designed Stellaris probes targeting RP11-350G8.5 have been conjugated with 5-carboxytetramethylrhodamine (TAMRA) dye (red signal). Representative pictures acquired with a DMI6000-AF6000 Leica (Wetzlar, Germany) fluorescence microscope at magnification ×63 are reported, followed by specific regions of interest (ROIs), which are represented as enlarged images. (F) Dose-response curves 24 hours after treatment with bortezomib in AMO-1 cells overexpressing RP11-350G8.5 (1-10 nM). Statistics were analyzed using multiple t -tests (cutoff ∗ P < .05, ∗∗ P < .01). (G) In vivo imaging of engrafted ABZB cells. A total of 5 × 10 6 ABZB cells, which previously underwent highly efficient transduction (multiplicity of infection = 1) of RP11-350G8.5 KO-GFP or the SCRAMBLE vectors, were subcutaneously inoculated in mice (n = 2 per group). Images of tumors were acquired when the tumoral masses became palpable (identified as DAY 1), and at the end of the experiment (DAY 16, when tumors reached 2 cm in diameter). Both DAY 1 and DAY 16 were set up by considering SCRAMBLE mice, because SCRAMBLE cells have been faster to generate tumoral masses, due to their higher proliferative rate, and to grow up to 2 cm in diameter, with respect to KO cells. Tumors appear as yellow high-density signals on the right flank of the mice. Pictures were obtained with the IVIS (Perkin Elmer) system. (H) Tumor growth as mean measurement ± standard deviation (SD) across mice groups (n = 2). (I) Photographs of excised tumors were captured by a digital camera. (J) Weights of excised tumors, reported as mean ± SD across mice groups. Statistics were analyzed using multiple t -tests (cutoff: ∗ P < .05).

    Journal: Blood

    Article Title: An unbiased lncRNA dropout CRISPR-Cas9 screen reveals RP11-350G8.5 as a novel therapeutic target for multiple myeloma ∗

    doi: 10.1182/blood.2023021991

    Figure Lengend Snippet: RP11-350G8.5 putative oncogenic role: in vitro validation and preliminary data from in vivo models. (A) Flow cytometric monitoring of GFP expression in ABZB cells transduced with a SCRAMBLE-GFP-CRISPR negative control vector, an RPL8 /KO-GFP-CRISPR positive control vector (selected from Project Score [37]), and 2 GFP-CRISPR constructs encoding for 2 pgRNAs targeting RP11-350G8.5. Gray curves represent the percentage of viable cells at day 0 (48 hours after lentiviral transduction), while colored curves represent the percentage of viable cells at day 20. Bars on the right represent the fold change in percentage of GFP-expressing cells 20 days after target depletion against day 0. (B) Evaluation of IL-6R RNA expression level through quantitative real time PCR (qRT-PCR) on ABZB after transduction with SCRAMBLE vector or KO of RP11-350G8.5 with pgRNA#1 or pgRNA#2 or with a vector overexpressing RP11-350G8.5 (UP). (Data are normalized to the expression of GAPDH.) Statistics were obtained using multiple t -tests, resulting in no significant (ns) differences, as per the reported P values. (C) Flow cytometric monitoring of GFP in JJN.3 and NCI-H929 MM transduced cells, and percentage of GFP-positive cells is reported by overlapping curves referred to day 20 (colored curves) against day 0 (light gray curves). (D) Validation of RP11-350G8.5 KO in nontumoral cells, performed as described for A and C. (E) Representative images of RNA-FISH analysis. Nuclei are counterstained with DAPI (blue signal), whereas C3-fluorescein–conjugated GAPDH (green signal) has been used as cytoplasmic marker. Customly designed Stellaris probes targeting RP11-350G8.5 have been conjugated with 5-carboxytetramethylrhodamine (TAMRA) dye (red signal). Representative pictures acquired with a DMI6000-AF6000 Leica (Wetzlar, Germany) fluorescence microscope at magnification ×63 are reported, followed by specific regions of interest (ROIs), which are represented as enlarged images. (F) Dose-response curves 24 hours after treatment with bortezomib in AMO-1 cells overexpressing RP11-350G8.5 (1-10 nM). Statistics were analyzed using multiple t -tests (cutoff ∗ P < .05, ∗∗ P < .01). (G) In vivo imaging of engrafted ABZB cells. A total of 5 × 10 6 ABZB cells, which previously underwent highly efficient transduction (multiplicity of infection = 1) of RP11-350G8.5 KO-GFP or the SCRAMBLE vectors, were subcutaneously inoculated in mice (n = 2 per group). Images of tumors were acquired when the tumoral masses became palpable (identified as DAY 1), and at the end of the experiment (DAY 16, when tumors reached 2 cm in diameter). Both DAY 1 and DAY 16 were set up by considering SCRAMBLE mice, because SCRAMBLE cells have been faster to generate tumoral masses, due to their higher proliferative rate, and to grow up to 2 cm in diameter, with respect to KO cells. Tumors appear as yellow high-density signals on the right flank of the mice. Pictures were obtained with the IVIS (Perkin Elmer) system. (H) Tumor growth as mean measurement ± standard deviation (SD) across mice groups (n = 2). (I) Photographs of excised tumors were captured by a digital camera. (J) Weights of excised tumors, reported as mean ± SD across mice groups. Statistics were analyzed using multiple t -tests (cutoff: ∗ P < .05).

    Article Snippet: The human paired-guide RNA (pgRNA) library pool ( ) (Addgene number 89640) was used to perform the CRISPR-Cas9 screens (supplemental Materials and methods; , and ).

    Techniques: In Vitro, Biomarker Discovery, In Vivo, Expressing, Transduction, CRISPR, Negative Control, Plasmid Preparation, Positive Control, Construct, RNA Expression, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Marker, Fluorescence, Microscopy, In Vivo Imaging, Infection, Standard Deviation

    Knockout of the CASP8AP2 gene. (a) Agarose gel electrophoresis of polymerase chain reaction (PCR) amplified CASP8AP2 locus in the parental and the 892–7 cell lines, and EMBOSS WATER sequence alignment of both alleles with the parental sequence. Guide RNA (gRNA) target sequence is highlighted. GAPDH-normalized qPCR analysis of CASP8AP2 messenger RNA (mRNA) abundance in parental and CASP8AP2 knockout clone, 892–7, using primers targeting the gRNA locus for amplification. (b) Caspase 8 activity in cell lysates of control (uninduced) and apoptosis-induced (induced) 892–7 and parental cells (**p < .001)

    Journal: Biotechnology and bioengineering

    Article Title: Knockout of the caspase 8-associated protein 2 gene improves recombinant protein expression in HEK293 cells through up-regulation of the cyclin-dependent kinase inhibitor 2A gene

    doi: 10.1002/bit.27561

    Figure Lengend Snippet: Knockout of the CASP8AP2 gene. (a) Agarose gel electrophoresis of polymerase chain reaction (PCR) amplified CASP8AP2 locus in the parental and the 892–7 cell lines, and EMBOSS WATER sequence alignment of both alleles with the parental sequence. Guide RNA (gRNA) target sequence is highlighted. GAPDH-normalized qPCR analysis of CASP8AP2 messenger RNA (mRNA) abundance in parental and CASP8AP2 knockout clone, 892–7, using primers targeting the gRNA locus for amplification. (b) Caspase 8 activity in cell lysates of control (uninduced) and apoptosis-induced (induced) 892–7 and parental cells (**p < .001)

    Article Snippet: CRISPR/Cas9 constructs Two plasmids encoding paired guide RNAs (gRNAs) targeting the 10th exon of the CASP8AP2 gene, designed by transOMIC Technologies, were used in this study.

    Techniques: Knock-Out, Agarose Gel Electrophoresis, Polymerase Chain Reaction, Amplification, Sequencing, Activity Assay, Control

    Luciferase and secreted alkaline phosphatase (SEAP) production from the parental cell line and the CASP8AP2 knockout clone, 892–7. (a) Luciferase enzymatic activity (***p < .0001). (b) GAPDH-normalized luciferase messenger RNA (mRNA) abundance determined using real-time quantitative polymerase chain reaction (** p < .001). (c) Luciferase expression by Western blot analysis. (d) Alkaline phosphatase activity in the cell culture supernatant of cells transfected with a plasmid encoding the SEAP gene (*p < .05). Results represent the average of at least three biological replicates, and error bars represent the standard deviation

    Journal: Biotechnology and bioengineering

    Article Title: Knockout of the caspase 8-associated protein 2 gene improves recombinant protein expression in HEK293 cells through up-regulation of the cyclin-dependent kinase inhibitor 2A gene

    doi: 10.1002/bit.27561

    Figure Lengend Snippet: Luciferase and secreted alkaline phosphatase (SEAP) production from the parental cell line and the CASP8AP2 knockout clone, 892–7. (a) Luciferase enzymatic activity (***p < .0001). (b) GAPDH-normalized luciferase messenger RNA (mRNA) abundance determined using real-time quantitative polymerase chain reaction (** p < .001). (c) Luciferase expression by Western blot analysis. (d) Alkaline phosphatase activity in the cell culture supernatant of cells transfected with a plasmid encoding the SEAP gene (*p < .05). Results represent the average of at least three biological replicates, and error bars represent the standard deviation

    Article Snippet: CRISPR/Cas9 constructs Two plasmids encoding paired guide RNAs (gRNAs) targeting the 10th exon of the CASP8AP2 gene, designed by transOMIC Technologies, were used in this study.

    Techniques: Luciferase, Knock-Out, Activity Assay, Real-time Polymerase Chain Reaction, Expressing, Western Blot, Cell Culture, Transfection, Plasmid Preparation, Standard Deviation

    The effect of CASP8AP2 knockout on cell cycle progression by FACS-based, propidium iodide cell cycle analysis. (a) Knockout clone. (b) Parental cells. (c) Percentages of cells in the G1, S, or G2/M in asynchronous cultures calculated using BD FACS DIVA software. Results represent the average of three analyses, and error bars represent the standard deviation (*p < .05). (d) Parental and 892–7 cells were treated with thymidine to synchronize the cell cycle. After release from the thymidine block, the cells were harvested at the indicated time points and cell cycle progression was analyzed using a cytometer

    Journal: Biotechnology and bioengineering

    Article Title: Knockout of the caspase 8-associated protein 2 gene improves recombinant protein expression in HEK293 cells through up-regulation of the cyclin-dependent kinase inhibitor 2A gene

    doi: 10.1002/bit.27561

    Figure Lengend Snippet: The effect of CASP8AP2 knockout on cell cycle progression by FACS-based, propidium iodide cell cycle analysis. (a) Knockout clone. (b) Parental cells. (c) Percentages of cells in the G1, S, or G2/M in asynchronous cultures calculated using BD FACS DIVA software. Results represent the average of three analyses, and error bars represent the standard deviation (*p < .05). (d) Parental and 892–7 cells were treated with thymidine to synchronize the cell cycle. After release from the thymidine block, the cells were harvested at the indicated time points and cell cycle progression was analyzed using a cytometer

    Article Snippet: CRISPR/Cas9 constructs Two plasmids encoding paired guide RNAs (gRNAs) targeting the 10th exon of the CASP8AP2 gene, designed by transOMIC Technologies, were used in this study.

    Techniques: Knock-Out, Cell Cycle Assay, Software, Standard Deviation, Blocking Assay, Cytometry

    Summary of raw and processed RNA-seq reads generated from CASP8AP2 mutant clone, 892–7, and parental RNA samples

    Journal: Biotechnology and bioengineering

    Article Title: Knockout of the caspase 8-associated protein 2 gene improves recombinant protein expression in HEK293 cells through up-regulation of the cyclin-dependent kinase inhibitor 2A gene

    doi: 10.1002/bit.27561

    Figure Lengend Snippet: Summary of raw and processed RNA-seq reads generated from CASP8AP2 mutant clone, 892–7, and parental RNA samples

    Article Snippet: CRISPR/Cas9 constructs Two plasmids encoding paired guide RNAs (gRNAs) targeting the 10th exon of the CASP8AP2 gene, designed by transOMIC Technologies, were used in this study.

    Techniques: Generated, Mutagenesis

    Pathway ranking based on Z-score following enrichment analysis of the differentially expressed genes between the CASP8AP2 knockout and the parental cell lines. Gray bars represent pathways involved in the cell cycle

    Journal: Biotechnology and bioengineering

    Article Title: Knockout of the caspase 8-associated protein 2 gene improves recombinant protein expression in HEK293 cells through up-regulation of the cyclin-dependent kinase inhibitor 2A gene

    doi: 10.1002/bit.27561

    Figure Lengend Snippet: Pathway ranking based on Z-score following enrichment analysis of the differentially expressed genes between the CASP8AP2 knockout and the parental cell lines. Gray bars represent pathways involved in the cell cycle

    Article Snippet: CRISPR/Cas9 constructs Two plasmids encoding paired guide RNAs (gRNAs) targeting the 10th exon of the CASP8AP2 gene, designed by transOMIC Technologies, were used in this study.

    Techniques: Knock-Out

    (a and b) Relative CDKN2A and HDAC10 expression in parental and CASP8AP2 knockout HEK293 cells determined in a GAPDH-normalized real-time quantitative polymerase chain reaction. (c) Effect of overexpressing CDKN2A on luciferase activity in parental (parental CDKN2A) and CASP8AP2 knockout (892–7 CDKN2A) HEK293 cells (*p < .05). (d) Effect of CDKN2A overexpression on cell growth of the parental cell line. Results represent the average of at least three biological replicates, with error bars representing standard deviation

    Journal: Biotechnology and bioengineering

    Article Title: Knockout of the caspase 8-associated protein 2 gene improves recombinant protein expression in HEK293 cells through up-regulation of the cyclin-dependent kinase inhibitor 2A gene

    doi: 10.1002/bit.27561

    Figure Lengend Snippet: (a and b) Relative CDKN2A and HDAC10 expression in parental and CASP8AP2 knockout HEK293 cells determined in a GAPDH-normalized real-time quantitative polymerase chain reaction. (c) Effect of overexpressing CDKN2A on luciferase activity in parental (parental CDKN2A) and CASP8AP2 knockout (892–7 CDKN2A) HEK293 cells (*p < .05). (d) Effect of CDKN2A overexpression on cell growth of the parental cell line. Results represent the average of at least three biological replicates, with error bars representing standard deviation

    Article Snippet: CRISPR/Cas9 constructs Two plasmids encoding paired guide RNAs (gRNAs) targeting the 10th exon of the CASP8AP2 gene, designed by transOMIC Technologies, were used in this study.

    Techniques: Expressing, Knock-Out, Real-time Polymerase Chain Reaction, Luciferase, Activity Assay, Over Expression, Standard Deviation

    Identification of the CDKN2A gene as an effector for recombinant protein expression in the CASP8AP2 deficient 892–7 clone

    Journal: Biotechnology and bioengineering

    Article Title: Knockout of the caspase 8-associated protein 2 gene improves recombinant protein expression in HEK293 cells through up-regulation of the cyclin-dependent kinase inhibitor 2A gene

    doi: 10.1002/bit.27561

    Figure Lengend Snippet: Identification of the CDKN2A gene as an effector for recombinant protein expression in the CASP8AP2 deficient 892–7 clone

    Article Snippet: CRISPR/Cas9 constructs Two plasmids encoding paired guide RNAs (gRNAs) targeting the 10th exon of the CASP8AP2 gene, designed by transOMIC Technologies, were used in this study.

    Techniques: Recombinant, Expressing