Journal: Nature biotechnology

Article Title: Quantitative analysis of RNA-protein interactions on a massively parallel array for mapping biophysical and evolutionary landscapes

doi: 10.1038/nbt.2880

Figure Lengend Snippet: (a) Steps for generating RNA tethered to DNA clusters on a high-throughput DNA sequencing flow cell. (b) Structure of the MS2 coat protein homodimer bound to the 19 nt hairpin RNA (PDB ID: 2BU1) . (c) Images of fluorescently labeled MS2 bound to RNA clusters at increasing concentrations of protein and at time points following perfusion of unlabeled MS2 competitor. Below, fitted sum of Gaussians used to assign fluorescence to clusters. Scale bars (white) represent 2.5 μ m . (d) Fluorescence decay of MS2 dissociating from clusters containing the consensus sequence (-5C) ( t 1/2 =8.39 minutes). (e) Fit binding curves to clusters labeled in panel ( c ). (f) The probability distribution of binding energies from all clusters with labeled variants; mean K d = 2.57 nM, 36.8 nM, and 415 nM for the -5C, -5U, and -5A variants, respectively. (g) Correlation between binding energies reported in the literature and measured on the RNA array (squares, Carey et al. , circles, Romaniuk et al. ). (Dashed line indicates our affinity measurement cutoff.)

Article Snippet: The MS2-dlFG mutant of the MS2 Coat Protein was cloned into a custom expression vector containing an N -terminal FLAG and SNAPtag (NEB) and a C -terminal 6xHis tag ( https://benchling.com/s/oYAOq4 ).

Techniques: High Throughput Screening Assay, DNA Sequencing, Labeling, Fluorescence, Sequencing, Binding Assay

Journal: Nature biotechnology

Article Title: Quantitative analysis of RNA-protein interactions on a massively parallel array for mapping biophysical and evolutionary landscapes

doi: 10.1038/nbt.2880

Figure Lengend Snippet: (a) Distribution of observed RNA variants by number of mutations. (b) Clusters measured per molecular variant as a function of mutation number. A median of ~11 clusters are observed for sequences with ≥4 mutations. Affinities for the consensus sequence come from N C =909,385 clusters. (c) Average − ΔΔG of point mutations per position. The − ΔΔG of alanine substitutions to the MS2 binding surface are shown in parentheses ( k B T ). Solid and dashed lines represent base and phosphate interactions, respectively. (d) Matrix of − ΔΔG for single and double mutants of the consensus sequence. Inset contains the m atrix of − ΔΔG for single and double mutants of the +1G variant. All energies are calculated relative to the consensus (-5C) sequence (arrow, − ΔΔG =0), and the number of quality-filtered double mutants in each matrix is indicated (M 2 ) . (e) Epistasis matrix derived from (d) allows de novo reconstruction of the hairpin structure.

Article Snippet: The MS2-dlFG mutant of the MS2 Coat Protein was cloned into a custom expression vector containing an N -terminal FLAG and SNAPtag (NEB) and a C -terminal 6xHis tag ( https://benchling.com/s/oYAOq4 ).

Techniques: Variant Assay, Mutagenesis, Sequencing, Binding Assay, Derivative Assay

Journal: Nature structural & molecular biology

Article Title: Spatiotemporal allele organization by allele-specific CRISPR live-cell imaging: SNP-CLING

doi: 10.1038/s41594-017-0015-3

Figure Lengend Snippet: (a) sgRNAs harboring internal protein-binding RNA-motifs (MS2, or PP7, or Puf1) direct non-catalytic dCas9 to each targeted locus. The corresponding RNA-binding proteins (RBP) MS2, or PP7, or PUM1, are fused to mVenus, or mCherry, or iRFP670, and fluorescently label up to three different loci. For allele-specific labelling, either the 2 nd or 3 rd nucleotide in the dCas9 PAM-motif 5′-NRG-3′ was substituted by a heterozygous SNP to a non-specific dCas9-motif (IUPAC code: ‘Y’ = ‘C’ or ‘T’; ‘H’ = ‘A’, ‘C’ or ‘T’), thereby preventing dCas9-binding to either the 129S1 or CAST alleles in mouse hybrid cells. Sanger sequencing of selected SNPs confirmed heterozygosity. (b) Allele-specific visualization of 129S1- Ypel4 (yellow = MS2-mVenus) and CAST- Ypel4 (red = PP7-mCherry) in male 129S1/CAST mESCs (scale bar = 5 μm, n = 4, 35 nuclei, dashed line = mESC nucleus, arrowheads = SNP-CLING foci of maternal and paternal Ypel4 alleles). (c) Three sgRNAs in MS2 and another set of three sgRNAs in PP7 targeted the CISTR-ACT locus (sgRNA-pool 1 and 2) in RPE-1 cells to elucidate specificity. (d) All measurable foci exhibited two-color co-localization indicating locus-specificity in female RPE-1 cells (edges of foci: < 1 voxel distance = < 50 nm 3 , scale bar = 500 nm, n = 4, 35 nuclei, arrowheads = CLING foci of CISTR-ACT ). (e) To address resolution, two sgRNAs pools targeted XIST (yellow = MS2-mVenus) and TSIX (red = PP7-mCherry), separated by a topological associated domain (TAD) boundary; genomic linear distance ~69 kb. (f) Distinct, non-co-localized signals occurred in 6 out of 10 cells between XIST and TSIX , with spatial displacements of ∼163-638 nm in all three dimensions in RPE-1 cells (scale bar = 100 nm, arrowheads = CLING foci of XIST and TSIX ).

Article Snippet: Flanking BbsI restriction sites were used to ligate sgRNAs with a backbone expressing either three MS2, or three PP7, or six Puf1 motifs (Addgene #68426, #68424), . mCherry or mVenus fluorescent proteins were expressed as fusion proteins with either MS2 or PP7 binding proteins (Addgene #68420), independent of dCas9 (Addgene #68416).

Techniques: Protein Binding, RNA Binding Assay, Binding Assay, Sequencing

Journal: Nature structural & molecular biology

Article Title: Spatiotemporal allele organization by allele-specific CRISPR live-cell imaging: SNP-CLING

doi: 10.1038/s41594-017-0015-3

Figure Lengend Snippet: (a) Inter -allelic distances of Hdac4 [CI = 2.31-3.59] on largest chromosome 1 compared to a locus on small chromosome 18 (44.21 Mb, [CI = 2.14-3.19 μm]) in female 129S1/CAST MEFs were similar (each 80 nuclei, scale bars = 5 μm, arrowheads = SNP-CLING foci of maternal and paternal Hdac4 alleles). (b) Inter -allelic distances between 129S1 and CAST alleles of loci on gene dense chromosomes (chr.7:99.55 Mb [CI = 2.48-3.96 μm], chr. 11 – Sox9 [CI = 1.82-2.75 μm]) or gene-poor chromosome 15 ( Cistr-act [CI = 2.05-3.03 μm]). Chr. 11 distances were different to chr. 7 (two-tailed Mann Whitney rank sum test, * p = 0.02, each 80 nuclei, arrowheads = SNP-CLING foci of maternal and paternal Sox9 alleles). (c) Inter -allelic distances of the SNP-CLING-labeled alleles on chr.7 (arrowheads: yellow = paternal MS2-mVenus, red = maternal PP7-mCherry) relative to inter -genic distances of a CLING-labeled locus (non-allele-specific) on chr.18 (arrowheads: purple = Puf1-PUM1-iRFP670, Pearson's r 2 = 0.71, significance of Pearson's: *** p < 0.0001, 80 nuclei).

Article Snippet: Flanking BbsI restriction sites were used to ligate sgRNAs with a backbone expressing either three MS2, or three PP7, or six Puf1 motifs (Addgene #68426, #68424), . mCherry or mVenus fluorescent proteins were expressed as fusion proteins with either MS2 or PP7 binding proteins (Addgene #68420), independent of dCas9 (Addgene #68416).

Techniques: Two Tailed Test, MANN-WHITNEY, Labeling

Journal: Nature structural & molecular biology

Article Title: Spatiotemporal allele organization by allele-specific CRISPR live-cell imaging: SNP-CLING

doi: 10.1038/s41594-017-0015-3

Figure Lengend Snippet: (a) Examples of merged images demonstrate positioning of the studied alleles (arrowheads: red = PP7-mCherry, yellow = MS2-mVenus), and the nucleoli (rRNA-GFP) in 129S1/CAST MEFs (scale bars = 5 μm, n = 5). (b) Similar allelic distances and distributions of loci to the nucleoli (means ± SD), between 129S1 and CAST alleles in 129S1/CAST MEFs (normalized to nuclei sizes, each combination > 80 nuclei). (c) Examples of loci-nuclear periphery measurements (see panel a, n = 6, arrowheads = SNP-CLING foci). (d) Allelic distances and distributions of loci to the nuclear periphery were similar between 129S1 or CAST alleles (each combination > 80 nuclei). Allelic distances of the studied loci to the nucleoli (means ± SD) were significantly smaller, than their distances to the nuclear periphery (two-tailed Mann Whitney rank sum testing, * p < 0.05, *** p < 0.0001). (e) Spearman correlations of all allelic distances to the nucleoli (n = 620 alleles, r 2 = 0.329, *** p < 0.0001) or to the nuclear periphery (r 2 = 0.512, *** p < 0.0001). 61 % of 129S1 or 64 % CAST inherited loci were closer than 1 μm to the nucleolus. 35 % of 129S1 or 30 % of CAST loci were closer than 1 μm to the periphery. (f) Number of nucleoli counted in human RPE-1 or MEFs was higher in MEFs (two-tailed Mann Whitney rank sum testing, *** p < 0.0001). (g) Similar inter -nucleoli distances in human RPE-1 or MEFs (Kruskal-Wallis multiple comparisons testing [not adjusted], ns = not significant, medians with 25 th to 75 th percentiles, min/max, and sample sizes [n] are depicted, RPE-1 CI lower = 3.02-3.31 μm, CI upper = 3.67-3.99 μm, MEFs CI lower = 3.06-3.35 μm, CI upper = 3.63-4.7 μm).

Article Snippet: Flanking BbsI restriction sites were used to ligate sgRNAs with a backbone expressing either three MS2, or three PP7, or six Puf1 motifs (Addgene #68426, #68424), . mCherry or mVenus fluorescent proteins were expressed as fusion proteins with either MS2 or PP7 binding proteins (Addgene #68420), independent of dCas9 (Addgene #68416).

Techniques: Two Tailed Test, MANN-WHITNEY

Journal: Nature structural & molecular biology

Article Title: Spatiotemporal allele organization by allele-specific CRISPR live-cell imaging: SNP-CLING

doi: 10.1038/s41594-017-0015-3

Figure Lengend Snippet: (a) Example of CLING-labeled FIRRE loci (arrowheads: red = PP7-mCherry, 108 nuclei) and their distances to the closest nucleolus (rRNA-GFP) in female RPE-1 cells (scale bars = 5 μm). Loci-nucleoli distances revealed that FIRRE was closer to the perinucleolar space than GAPDH (107 nuclei, two-tailed Mann-Whitney rank sum test, *** p < 0.0001). (b) Example of maternal and paternal Firre alleles (arrowheads) in female 129S1 (yellow = MS2-mVenus), CAST (red = PP7-mCherry) MEFs. Firre's allelic distances to the nucleoli (129S1 CI = 0.77-1.32 μm, CAST CI = 0.81-1.38 μm) or to the nuclear periphery (80 nuclei, means ± SD, 129S1 CI = 1.38-1.84 μm, CAST CI = 1.48-1.89 μm). Firre was closer to the nucleoli than to the nuclear periphery (two-tailed Mann Whitney rank sum test, *** p < 0.0001). (c) Scheme of interacting loci between two non-homologous chromosomes in spatial proximity, targeted by SNP-CLING or CLING. (d) SNP-CLING of maternal 129S1-derived Firre (yellow = MS2-mVenus) with either Ypel4 -129S1 or Ypel4 -CAST (red = PP7-mCherry) in male mESCs (n = 8, arrowheads = SNP-CLING foci). (e) Allele-biased interaction of Firre with the paternal Ypel4 -CAST locus (** p = 0.0012, Χ 2 -test, each quantification > 130 nuclei, n = 5) in male 129S1/CAST mESCs.

Article Snippet: Flanking BbsI restriction sites were used to ligate sgRNAs with a backbone expressing either three MS2, or three PP7, or six Puf1 motifs (Addgene #68426, #68424), . mCherry or mVenus fluorescent proteins were expressed as fusion proteins with either MS2 or PP7 binding proteins (Addgene #68420), independent of dCas9 (Addgene #68416).

Techniques: Labeling, Two Tailed Test, MANN-WHITNEY, Derivative Assay

Journal: The Journal of Clinical Investigation

Article Title: APC-activated long noncoding RNA inhibits colorectal carcinoma pathogenesis through reduction of exosome production

doi: 10.1172/JCI122478

Figure Lengend Snippet: Expression of APC in the indicated cell lines transfected with control or WT APC vector, as measured by qRT-PCR (A) and Western blotting (B). (C) Number of altered lncRNAs in the indicated cells examined in 2 independently repeated lncRNA microarray tests. (D) qRT-PCR verification of lncRNAs potentially regulated by APC. (E) Expression of lncRNA-APC1 was detected by FISH. Scale bars: 20 μm. (F) Relative expression of lncRNA-APC1 in paired CRC primary tumor tissues and nontumor colonic tissues (n = 30). (G) Kaplan-Meier survival analysis of patients with CRC (n = 110) according to lncRNA-APC1 expression (cutoff value is the median). Experiments in F and G were repeated twice with similar results. Data in A, E, and F represent the mean ± SD of 3 separate experiments. **P < 0.01, ***P < 0.001, and ****P < 0.0001, by independent Student’s t test (A and F) or log-rank test (G). NC, negative control.

Article Snippet: pSL-MS2-12x (Addgene) was digested with BamH I and Not I, and the MS2-12x fragment was subcloned into pcDNA3.1, pcDNA3.1-lncRNA-APC1-WT, and pcDNA3.1-lncRNA-APC1-Mut (Rab5b), which were named pcDNA3.1-MS2, pcDNA3.1-MS2-lncRNA-APC1-WT, and pcDNA3.1-MS2-lncRNA-APC1-Mut (Rab5b), respectively.

Techniques: Expressing, Transfection, Plasmid Preparation, Quantitative RT-PCR, Western Blot, Microarray, Negative Control

Journal: The Journal of Clinical Investigation

Article Title: APC-activated long noncoding RNA inhibits colorectal carcinoma pathogenesis through reduction of exosome production

doi: 10.1172/JCI122478

Figure Lengend Snippet: (A) Luciferase activity of pGL3-lncRNA-APC1 promoter luciferase in DLD-1 cells. (B and C) Luciferase activity of the indicated pGL3-lncRNA-APC1 promoter luciferase vectors. (D) PPARα was efficiently knocked down by siRNA, as detected by Western blotting. (E) qRT-PCR analysis of lncRNA-APC1 expression induced by PPARα knockdown. (F) Luciferase activity in the indicated cells cotransfected with WT or PPARα binding motif deletion (Mut-del) pGL3-lncRNA-APC1 promoter luciferase. (G) Ectopic expression of PPARα was substantially increased in DLD-1 cells, as shown by Western blotting. (H) Luciferase activity of the lncRNA-APC1 promoter cotransfected with APC and/or the PPARα construct. (I) ChIP analysis to detect the enrichment of PPARα on the promoter of lncRNA-APC1. All luciferase data were normalized to Renilla luciferase activity. Data represent the mean ± SD of 3 separate experiments. All experiments were repeated at least 3 times. *P < 0.05, **P < 0.01, and ***P < 0.001, and ****P < 0.0001, by independent Student’s t test (A, F, and I) or 1-way ANOVA (C, E, and H).

Article Snippet: pSL-MS2-12x (Addgene) was digested with BamH I and Not I, and the MS2-12x fragment was subcloned into pcDNA3.1, pcDNA3.1-lncRNA-APC1-WT, and pcDNA3.1-lncRNA-APC1-Mut (Rab5b), which were named pcDNA3.1-MS2, pcDNA3.1-MS2-lncRNA-APC1-WT, and pcDNA3.1-MS2-lncRNA-APC1-Mut (Rab5b), respectively.

Techniques: Luciferase, Activity Assay, Western Blot, Quantitative RT-PCR, Expressing, Binding Assay, Construct

Journal: The Journal of Clinical Investigation

Article Title: APC-activated long noncoding RNA inhibits colorectal carcinoma pathogenesis through reduction of exosome production

doi: 10.1172/JCI122478

Figure Lengend Snippet: (A) Proliferation rate of empty vector– or lncRNA-APC1–transfected cells as determined by CCK8 assay (P < 0.05). (B) Representative images of decreased foci formation. (C) Images of xenograft tumors formed in nude mice. (D) Representative images of H&E-stained sections from metastatic nodules in the lung (original magnification, ×100). (E) Representative images of hepatic and splenic tissue in a nude mouse metastasis model. Black arrow indicates CRC cells; green arrow indicates liver tissue; blue arrow indicates splenic tissue (original magnification, ×100). Data represent the mean ± SD of 3 independent experiments. Scale bars: 50 μm. **P < 0.01, ***P < 0.001, and ****P < 0.0001, by independent Student’s t test.

Article Snippet: pSL-MS2-12x (Addgene) was digested with BamH I and Not I, and the MS2-12x fragment was subcloned into pcDNA3.1, pcDNA3.1-lncRNA-APC1-WT, and pcDNA3.1-lncRNA-APC1-Mut (Rab5b), which were named pcDNA3.1-MS2, pcDNA3.1-MS2-lncRNA-APC1-WT, and pcDNA3.1-MS2-lncRNA-APC1-Mut (Rab5b), respectively.

Techniques: Plasmid Preparation, Transfection, CCK-8 Assay, Staining

Journal: The Journal of Clinical Investigation

Article Title: APC-activated long noncoding RNA inhibits colorectal carcinoma pathogenesis through reduction of exosome production

doi: 10.1172/JCI122478

Figure Lengend Snippet: (A) APC expression in the DLD-1 stable cell line. (B) Relative expression of lncRNA-APC1 in DLD-1 cells transfected with shRNAs specific for silencing lncRNA-APC1. (C) Images of xenograft tumors formed in nude mice. (D) Representative images of H&E-stained sections from metastatic nodules in the lung (original magnification, ×100). Scale bars: 50 μm. Data represent the mean ± SD of 3 independent experiments. *P < 0.05, **P < 0.01, and ****P < 0.0001, by 1-way ANOVA.

Article Snippet: pSL-MS2-12x (Addgene) was digested with BamH I and Not I, and the MS2-12x fragment was subcloned into pcDNA3.1, pcDNA3.1-lncRNA-APC1-WT, and pcDNA3.1-lncRNA-APC1-Mut (Rab5b), which were named pcDNA3.1-MS2, pcDNA3.1-MS2-lncRNA-APC1-WT, and pcDNA3.1-MS2-lncRNA-APC1-Mut (Rab5b), respectively.

Techniques: Expressing, Stable Transfection, Transfection, Staining

Journal: The Journal of Clinical Investigation

Article Title: APC-activated long noncoding RNA inhibits colorectal carcinoma pathogenesis through reduction of exosome production

doi: 10.1172/JCI122478

Figure Lengend Snippet: (A) Relative expression of angiogenesis-associated factors in cells stably overexpressing lncRNA-APC1 or in control cells. (B) Western blot analysis of exosome markers. (C) Representative images of exosomes by electron microscopic detection. Scale bars: 200 nm. (D) Size distribution of exosomes analyzed by NanoSight. Relative fold changes in protein concentrations of exosome lysates as determined by (E) bicinchoninic acid (BCA) assay or (F) NanoSight analysis. (G) Relative fold changes in protein concentrations of exosome lysates as revealed by BCA assay. All experiments were repeated at least 3 times. Data represent the mean ± SD of 3 independent experiments. ***P < 0.001 and ****P < 0.0001, by independent Student’s t test (E and F) or 1-way ANOVA (G).

Article Snippet: pSL-MS2-12x (Addgene) was digested with BamH I and Not I, and the MS2-12x fragment was subcloned into pcDNA3.1, pcDNA3.1-lncRNA-APC1-WT, and pcDNA3.1-lncRNA-APC1-Mut (Rab5b), which were named pcDNA3.1-MS2, pcDNA3.1-MS2-lncRNA-APC1-WT, and pcDNA3.1-MS2-lncRNA-APC1-Mut (Rab5b), respectively.

Techniques: Expressing, Stable Transfection, Western Blot, BIA-KA

Journal: The Journal of Clinical Investigation

Article Title: APC-activated long noncoding RNA inhibits colorectal carcinoma pathogenesis through reduction of exosome production

doi: 10.1172/JCI122478

Figure Lengend Snippet: (A) Relative expression of Rab genes as determined by qRT-PCR. (B) Rab5b expression in the indicated lncRNA-APC1 stable cells or control cells as detected by Western blotting. Levels of Rab5b mRNA (C) and Rab5b protein (D) in the indicated cells. (E) Rab5b was efficiently knocked down by specific shRNAs. (F) Relative fold change in the protein concentration of exosome lysates as determined by BCA assay. (G) Images of the xenograft tumors formed in nude mice by injecting Rab5b–stably silenced or control cells. Data represent the mean ± SD of 3 independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001, by independent Student’s t test (A) or 1-way ANOVA (C, F, and G).

Article Snippet: pSL-MS2-12x (Addgene) was digested with BamH I and Not I, and the MS2-12x fragment was subcloned into pcDNA3.1, pcDNA3.1-lncRNA-APC1-WT, and pcDNA3.1-lncRNA-APC1-Mut (Rab5b), which were named pcDNA3.1-MS2, pcDNA3.1-MS2-lncRNA-APC1-WT, and pcDNA3.1-MS2-lncRNA-APC1-Mut (Rab5b), respectively.

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Protein Concentration, BIA-KA, Stable Transfection

Journal: The Journal of Clinical Investigation

Article Title: APC-activated long noncoding RNA inhibits colorectal carcinoma pathogenesis through reduction of exosome production

doi: 10.1172/JCI122478

Figure Lengend Snippet: (A) Cell proliferation rate induced by lncRNA-APC1 silencing and/or Rab5b knockdown as determined by CCK8 assay. (B) Representative images of foci formation. (C) Representative images of the Transwell invasion assay. Scale bars: 100 μm. Data represent the mean ± SD of 3 independent experiments. **P < 0.01, ***P < 0.001, and ****P < 0.0001, by 1-way ANOVA.

Article Snippet: pSL-MS2-12x (Addgene) was digested with BamH I and Not I, and the MS2-12x fragment was subcloned into pcDNA3.1, pcDNA3.1-lncRNA-APC1-WT, and pcDNA3.1-lncRNA-APC1-Mut (Rab5b), which were named pcDNA3.1-MS2, pcDNA3.1-MS2-lncRNA-APC1-WT, and pcDNA3.1-MS2-lncRNA-APC1-Mut (Rab5b), respectively.

Techniques: CCK-8 Assay, Transwell Invasion Assay

Journal: The Journal of Clinical Investigation

Article Title: APC-activated long noncoding RNA inhibits colorectal carcinoma pathogenesis through reduction of exosome production

doi: 10.1172/JCI122478

Figure Lengend Snippet: (A) Regions of putative binding between Rab5b mRNA (query) and lncRNA-APC1 (subject). (B) SW480 and DLD-1 cell lysates were incubated with biotin-labeled WT or mutant type (Mut) lncRNA-APC1. After pulldown, mRNA was extracted and measured by qRT-PCR. (C) Model of RIP assay. (D) RIP-derived RNA was examined by qRT-PCR. The levels of the qRT-PCR products were normalized relative to input RNA and IgG control. (E) The stability of Rab5b mRNA and Actb mRNA was measured by qRT-PCR relative to t0 after blocking new RNA synthesis with α-amanitin and normalized to 18S rRNA. Data represent the mean ± SD of 3 independent experiments. **P < 0.01, ***P < 0.001, and ****P < 0.0001, by 1-way ANOVA.

Article Snippet: pSL-MS2-12x (Addgene) was digested with BamH I and Not I, and the MS2-12x fragment was subcloned into pcDNA3.1, pcDNA3.1-lncRNA-APC1-WT, and pcDNA3.1-lncRNA-APC1-Mut (Rab5b), which were named pcDNA3.1-MS2, pcDNA3.1-MS2-lncRNA-APC1-WT, and pcDNA3.1-MS2-lncRNA-APC1-Mut (Rab5b), respectively.

Techniques: Binding Assay, Incubation, Labeling, Mutagenesis, Quantitative RT-PCR, Derivative Assay, Blocking Assay

Journal: The Journal of Clinical Investigation

Article Title: APC-activated long noncoding RNA inhibits colorectal carcinoma pathogenesis through reduction of exosome production

doi: 10.1172/JCI122478

Figure Lengend Snippet: (A) Function (left) and pathway (right) enrichment analysis of the results from the gene expression profile microarray. (B) Relative expression of the indicated genes measured by qRT-PCR. (C) Western blot analysis shows that the MAPK pathway in HUVECs was activated by exosomes derived from lncRNA-APC1–silenced HCT116 cells. p-p44/42, phosphorylated p44/42; p-p38, phosphorylated p38. (D) Capillary tube formation (top) and Transwell invasion assay (bottom) of HUVECs treated with the indicated exosomes (P < 0.05, by 1-way ANOVA). Scale bars: 100 μm. The capillary tube formation experiment was performed at least 3 times, independently of the assay in Figure 7D. Data represent the mean ± SD of 3 independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.001, by independent Student’s t test (B) or 1-way ANOVA (D).

Article Snippet: pSL-MS2-12x (Addgene) was digested with BamH I and Not I, and the MS2-12x fragment was subcloned into pcDNA3.1, pcDNA3.1-lncRNA-APC1-WT, and pcDNA3.1-lncRNA-APC1-Mut (Rab5b), which were named pcDNA3.1-MS2, pcDNA3.1-MS2-lncRNA-APC1-WT, and pcDNA3.1-MS2-lncRNA-APC1-Mut (Rab5b), respectively.

Techniques: Expressing, Microarray, Quantitative RT-PCR, Western Blot, Derivative Assay, Transwell Invasion Assay

Journal: The Journal of Clinical Investigation

Article Title: APC-activated long noncoding RNA inhibits colorectal carcinoma pathogenesis through reduction of exosome production

doi: 10.1172/JCI122478

Figure Lengend Snippet: (A) Wnt1 expression in the indicated CRC cell–derived exosomes. (B) Wnt1 expression was effectively knocked down by specific siRNA-2 and siRNA-3. (C) Expression of lncRNA-APC1 detected by qRT-PCR. (D) Representative images of decreased foci formation induced by Wnt1-silencing exosomes. (E) Representative images of Transwell invasion assay. Scale bars: 50 μm. Data represent the mean ± SD of 3 independent experiments. **P < 0.01, ***P < 0.001, and ****P < 0.001, by 1-way ANOVA.

Article Snippet: pSL-MS2-12x (Addgene) was digested with BamH I and Not I, and the MS2-12x fragment was subcloned into pcDNA3.1, pcDNA3.1-lncRNA-APC1-WT, and pcDNA3.1-lncRNA-APC1-Mut (Rab5b), which were named pcDNA3.1-MS2, pcDNA3.1-MS2-lncRNA-APC1-WT, and pcDNA3.1-MS2-lncRNA-APC1-Mut (Rab5b), respectively.

Techniques: Expressing, Derivative Assay, Quantitative RT-PCR, Transwell Invasion Assay

Journal: The Journal of Clinical Investigation

Article Title: APC-activated long noncoding RNA inhibits colorectal carcinoma pathogenesis through reduction of exosome production

doi: 10.1172/JCI122478

Figure Lengend Snippet: lncRNA-APC1, an important mediator of APC, inhibits the pathogenesis and/or angiogenesis of CRC through directly reducing Rab5b mRNA stability and exosome production in CRC cells.

Article Snippet: pSL-MS2-12x (Addgene) was digested with BamH I and Not I, and the MS2-12x fragment was subcloned into pcDNA3.1, pcDNA3.1-lncRNA-APC1-WT, and pcDNA3.1-lncRNA-APC1-Mut (Rab5b), which were named pcDNA3.1-MS2, pcDNA3.1-MS2-lncRNA-APC1-WT, and pcDNA3.1-MS2-lncRNA-APC1-Mut (Rab5b), respectively.

Techniques:

Journal: Molecular Therapy. Nucleic Acids

Article Title: Transient Retrovirus-Based CRISPR/Cas9 All-in-One Particles for Efficient, Targeted Gene Knockout

doi: 10.1016/j.omtn.2018.09.006

Figure Lengend Snippet: Design of MLV-Based Gag.MS2.CRISPR/Cas9 All-in-One Chimera (A) Schematic illustration of Gag.MS2.CRISPR/Cas9 all-in-one particles. Chimeric Gag.MS2 proteins bind to non-viral RNAs containing two copies of a high-affinity MS2 target site (TS) hairpin structure. Hypothetically, redirection of the MLV-based packaging machinery should now allow specific packaging of different RNA species. In the case of CRISPR/Cas9, TS-containing Cas9 mRNA and Pol III-driven sgRNA transcripts should be co-packaged into one Gag.MS2 particle. (B) Design of the Gag.MS2 expression construct. The MLV-based wild-type Gag-Pol expression construct is depicted on top. In this plasmid, expression of Gag and Gag-Pol precursor proteins is initiated by enhancer and promoter sequences of the cytomegalovirus (CMV) and terminated by the bovine growth hormone poly(A) signal (pA). During particle maturation, Gag and Gag-Pol proteins are further processed into matrix (MA), p12, capsid (CA), NC, protease (PR), reverse transcriptase (RT), and integrase (IN) subunits. To generate the Gag.MS2 expression plasmids, coding sequences for Gag and Pol were separated on two CMV-driven plasmids. Subsequently, NC was replaced by a genetically fused MS2 heterodimer. All viral protease sites separating Gag and Pol subunits were maintained. (C) SpCas9 expression plasmids with or without 2 MS2 TS copies. SpCas9 was co-expressed with EGFP, and separation of both proteins was achieved via the P2A cleavage site from porcine teschovirus. PRE, woodchuck hepatitis virus post-transcriptional regulatory element. (D) Non-viral sgRNA expression plasmids targeting Tet2 , Tp53 , PTEN , TP53 , CXCR4 , or Renilla luciferase (Luc). sgRNAs with or without TS hairpins were generated. Two TS hairpins were either incorporated (TS.inc) into or cloned adjacent (TS.adj) to the sgRNA scaffold. To control for transfection efficiencies, a CMV-driven red-fluorescent DsRedexp expression cassette was included. hU6, human RNA polymerase III U6 promoter. (E) Detailed overview of TS.inc and TS.adj expression cassettes. The scaffold sequence of TS.inc is depicted on top, and the scaffold sequence of TS.adj is at the bottom. Both sgRNAs contain a 105 bp long stuffer sequence flanked by BsmBI restriction sites (red). The design of the TS.inc scaffold was adapted from Konermann et al., and positioning of the two TS hairpins in the TS.adj constructs was based on the work of Mali et al. with differences in TS linker and spacer sequences. The positions of the two TS hairpins (TS 1 and TS 2) are marked in blue. The arrows point to the cutting sites of BsmBI. Expression of the sgRNAs is terminated by the TTTTTT motif.

Article Snippet: After staining all blotted proteins with Ponceau S (Sigma-Aldrich), the membrane was successively probed with a rabbit polyclonal anti-enterobacteriophage MS2 coat protein antibody (1:5,000) (Merck Millipore, Darmstadt, Germany) and a monoclonal mouse purified anti-CRISPR (Cas9) antibody (1:1,500) (BioLegend, Koblenz, Germany) in Tris-buffered saline with 0.05% Tween and 3% milk powder at 4°C overnight (both Roth, Karlsruhe, Germany).

Techniques: CRISPR, Expressing, Construct, Plasmid Preparation, Reverse Transcription, Virus, Luciferase, Generated, Clone Assay, Control, Transfection, Sequencing

Journal: Molecular Therapy. Nucleic Acids

Article Title: Transient Retrovirus-Based CRISPR/Cas9 All-in-One Particles for Efficient, Targeted Gene Knockout

doi: 10.1016/j.omtn.2018.09.006

Figure Lengend Snippet: Individual Transfer of CRISPR/Cas9 RNAs by Gag.MS2 Particles (A) Highly efficient delivery of SpCas9.TS mRNA into target cells. Gag.MS2.SpCas9.TS particles were used to transduce NIH3T3-based RFP657.Tet2+sgRNA.Tet2 reporter cells (see the scheme above the graph) with the depicted supernatant volumes. The percentage of RFP657-negative cells was determined 6 days post-transduction. The results of 8 independent supernatants are shown. (B) Inefficient transfer of sgRNAs Tet2.TS.inc and Tet2.TS.adj by Gag.MS2 particles. RFP657.Tet2+SpCas9 NIH3T3 reporter cells were transduced with Tet2.TS.inc or Tet2.TS.adj Gag.MS2 particles. RFP657 knockout was determined by flow cytometry 6 days post-transduction. The results of 10 (Tet2.TS.inc) to 13 (Tet2.TS.adj) independently packaged supernatants are shown. (C) Co-transduction of separately packaged SpCas9.TS mRNA and sgRNA.TS resulted in poor RFP657 knockout rates. NIH3T3-based RFP657.Tet2 reporter cells were transduced with 6–8 individually packaged SpCas9.TS and Tet2.TS.inc or Tet2.TS.adj Gag.MS2 supernatants from (A) and (B) at the depicted volume ratios. Flow cytometry was performed 6 days post-transduction.

Article Snippet: After staining all blotted proteins with Ponceau S (Sigma-Aldrich), the membrane was successively probed with a rabbit polyclonal anti-enterobacteriophage MS2 coat protein antibody (1:5,000) (Merck Millipore, Darmstadt, Germany) and a monoclonal mouse purified anti-CRISPR (Cas9) antibody (1:1,500) (BioLegend, Koblenz, Germany) in Tris-buffered saline with 0.05% Tween and 3% milk powder at 4°C overnight (both Roth, Karlsruhe, Germany).

Techniques: CRISPR, Transduction, Knock-Out, Flow Cytometry

Journal: Molecular Therapy. Nucleic Acids

Article Title: Transient Retrovirus-Based CRISPR/Cas9 All-in-One Particles for Efficient, Targeted Gene Knockout

doi: 10.1016/j.omtn.2018.09.006

Figure Lengend Snippet: Efficient RFP657 Knockout by Gag.MS2.CRISPR/Cas9 All-in-One Particles SpCas9, Tet2 sgRNA, Gag.MS2, Pol, and VSVg expression plasmids were co-transfected into the HEK293T packaging cell line. The resulting supernatants SpCas9+Tet2, SpCas9.TS+Tet2.TS.inc, or SpCas9.TS+Tet2.TS.adj were harvested and concentrated 50-fold. Transfection efficiencies were determined for all produced supernatants (see also A and S4B). (A and B) Efficient delivery of CRISPR/Cas9 RNA components depends on the presence of the TS hairpin dimer. Supernatants from HEK293T cells that exhibited transfection efficiencies <50% were used to transduce NIH3T3-based (A) or HT1080-based (B) RFP657.Tet2 reporter cells. The graphs show RFP657 knockout rates mediated by 100 μL supernatant for the individual experiments 5–6 days post-transduction. Results from 4–7 independently generated supernatants are displayed (SpCas9+Tet2 = 6 supernatants, SpCas9.TS+Tet2.TS.inc = 7 supernatants, and SpCas9.TS+Tet2.TS.adj = 4 supernatants) (see also Figure S4 A). (C and D) Non-specific packaging of CRISPR/Cas9 RNAs and/or RNPs into Gag.MS2 particles. Experiments were similar to those shown in (A) and (B). However, NIH3T3 (C) and HT1080 (D) RFP657.Tet2 reporter cells were transduced with supernatants that were obtained from transfections with efficiencies >50%. The depicted results were generated with 100 μL of 10–12 individually packaged supernatants (SpCas9+Tet2 = 10 supernatants, SpCas9.TS+Tet2.TS.inc = 10 supernatants, and SpCas9.TS+Tet2.TS.adj = 12 supernatants) (see also Figure S4 B). The graph depicted in (D) also displays RFP657 knockout rates induced by different volumes of 50-fold-concentrated integrating RIT.CRISPR/Cas9.Tet2 all-in-one supernatants. The respective MOIs are given in the gray box. Each data point represents an individually generated supernatant (n = 4) with an average functional titer of 7.2 × 10 7 t.u./mL.

Article Snippet: After staining all blotted proteins with Ponceau S (Sigma-Aldrich), the membrane was successively probed with a rabbit polyclonal anti-enterobacteriophage MS2 coat protein antibody (1:5,000) (Merck Millipore, Darmstadt, Germany) and a monoclonal mouse purified anti-CRISPR (Cas9) antibody (1:1,500) (BioLegend, Koblenz, Germany) in Tris-buffered saline with 0.05% Tween and 3% milk powder at 4°C overnight (both Roth, Karlsruhe, Germany).

Techniques: Knock-Out, CRISPR, Expressing, Transfection, Produced, Transduction, Generated, Functional Assay

Journal: Molecular Therapy. Nucleic Acids

Article Title: Transient Retrovirus-Based CRISPR/Cas9 All-in-One Particles for Efficient, Targeted Gene Knockout

doi: 10.1016/j.omtn.2018.09.006

Figure Lengend Snippet: Characterization of Gag.MS2.CRISPR/Cas9 Particles (A) SpCas9 and SpCas9.TS mRNA content of individually packaged Gag.MS2 particles. Absolute mRNA copies per 50 μL supernatant are shown. (B) Tet2 sgRNA quantification. The differences of incorporated Tet2 (no TS), Tet2.TS.inc, or Tet2.TS.adj sgRNAs are shown. The graph depicts the sgRNA copies per 50 μL. Absolute amounts were calculated with the help of individual standards. (C) Gag.MS2.CRISPR/Cas9 supernatants contain SpCas9 protein. SpCas9, SpCas9.TS, SpCas9.TS+Tet2.TS.inc (TS.inc), and SpCas9.TS+Tet2.TS.adj (TS.adj) Gag.MS2 supernatants were subjected to immunoblot analysis, and membranes were successively stained with Ponceau S for total, SpCas9 (163 kDa), and MS2 dimer (27 kDa) proteins. The 82 kDa band on the MS2 blot represents the immature Gag.MS2 precursor polyprotein. (D) Quantification of SpCas9 protein by ELISA within different particle types. Each data point represents one independent supernatant. (E) Chimeric Gag.MS2.CRISPR/Cas9 all-in-one particles do not integrate. The genomic DNA of transduced cells was harvested 10 days post-transduction and analyzed for the occurrence of stable integration events. The graph depicts the determined mean vector copy numbers (VCNs) of two independently generated supernatants. An integrating RIT.CRISPR/Cas9.Tet2 all-in-one vector served as a positive control (average transduction rate: 14% EGFP-positive cells). Each data point represents the mean value of 3 independently determined C t values (technical replicates).

Article Snippet: After staining all blotted proteins with Ponceau S (Sigma-Aldrich), the membrane was successively probed with a rabbit polyclonal anti-enterobacteriophage MS2 coat protein antibody (1:5,000) (Merck Millipore, Darmstadt, Germany) and a monoclonal mouse purified anti-CRISPR (Cas9) antibody (1:1,500) (BioLegend, Koblenz, Germany) in Tris-buffered saline with 0.05% Tween and 3% milk powder at 4°C overnight (both Roth, Karlsruhe, Germany).

Techniques: CRISPR, Western Blot, Staining, Enzyme-linked Immunosorbent Assay, Transduction, Plasmid Preparation, Generated, Positive Control

Journal: Molecular Therapy. Nucleic Acids

Article Title: Transient Retrovirus-Based CRISPR/Cas9 All-in-One Particles for Efficient, Targeted Gene Knockout

doi: 10.1016/j.omtn.2018.09.006

Figure Lengend Snippet: Detailed Analysis of Gag.MS2.CRISPR/Cas9 All-in-One Particles

Article Snippet: After staining all blotted proteins with Ponceau S (Sigma-Aldrich), the membrane was successively probed with a rabbit polyclonal anti-enterobacteriophage MS2 coat protein antibody (1:5,000) (Merck Millipore, Darmstadt, Germany) and a monoclonal mouse purified anti-CRISPR (Cas9) antibody (1:1,500) (BioLegend, Koblenz, Germany) in Tris-buffered saline with 0.05% Tween and 3% milk powder at 4°C overnight (both Roth, Karlsruhe, Germany).

Techniques: CRISPR, Knock-Out, Functional Assay

Journal: Molecular Therapy. Nucleic Acids

Article Title: Transient Retrovirus-Based CRISPR/Cas9 All-in-One Particles for Efficient, Targeted Gene Knockout

doi: 10.1016/j.omtn.2018.09.006

Figure Lengend Snippet: Stable and High-Dose SpCas9 Expression Is Cytotoxic (A) Reduced cell numbers in RIT.SpCas9-treated cultures. NIH3T3 cells were transduced with integrating RIT.EGFP or RIT.SpCas9 particles or non-integrating Gag.MS2.SpCas9.TS particles. Based on titration via real-time qRT-PCR, RIT.EGFP and RIT.SpCas9 supernatants were adjusted before transduction (see also Figure S6 D). Gag.MS2.SpCas9.TS supernatants were not adjusted. Cell counts were determined 4 days post-transduction. Non-treated NIH3T3 cells served as Mock control. (B) Prolonged and high-dose SpCas9 expression decreases the metabolic activity of NIH3T3 cells. To assess the metabolic activity, transduced cells of (A) were subjected to the colorimetric MTS cell proliferation and cytotoxicity assay. The graph shows the absorbance (490 nm) of purple formazan, which originated from the reduction of MTS by cellular NAD(P)H. Results are shown as mean ± SD. (C) High-dose SpCas9 expression induces apoptosis. Transduced cells of (A) were co-stained with Annexin V and propidium iodide (PI) and analyzed for the occurrence of apoptotic (Annexin V + ) and dead (PI + or Annexin V + /PI + ) cells. The left graph depicts the total percentage of Annexin V + cells in the respective samples. The right graph shows Annexin V + , PI + and Annexin V + /PI + cells of non-transduced Mock cells and cultures that were transduced with RIT.SpCas9. The error bars in the right graph indicate the SD of the mean. (D) High-dose SpCas9 expression resulted in a substantial G0/G1 cell-cycle arrest. NIH3T3 cells were freshly transduced with the depicted supernatants and volumes. Cultures were subjected to cell-cycle analysis 6 days post-transduction. The percentages of cells in G0/G1 and G2/M of transduced cultures were shown relative to the respective phase of non-transduced Mock cultures. Results depicted in (A)–(C) were generated with 2–3 independently packaged supernatants (RIT.EGFP = 2–3 supernatants, RIT.SpCas9 = 3 supernatants, and Gag.MS2.SpCas9.TS = 3 supernatants). Each data point presented in (D) was obtained from independently packaged supernatants (n = 3 biological replicates).

Article Snippet: After staining all blotted proteins with Ponceau S (Sigma-Aldrich), the membrane was successively probed with a rabbit polyclonal anti-enterobacteriophage MS2 coat protein antibody (1:5,000) (Merck Millipore, Darmstadt, Germany) and a monoclonal mouse purified anti-CRISPR (Cas9) antibody (1:1,500) (BioLegend, Koblenz, Germany) in Tris-buffered saline with 0.05% Tween and 3% milk powder at 4°C overnight (both Roth, Karlsruhe, Germany).

Techniques: Expressing, Transduction, Titration, Quantitative RT-PCR, Control, Activity Assay, Cytotoxicity Assay, Staining, Cell Cycle Assay, Generated

Journal: Molecular Therapy. Nucleic Acids

Article Title: Transient Retrovirus-Based CRISPR/Cas9 All-in-One Particles for Efficient, Targeted Gene Knockout

doi: 10.1016/j.omtn.2018.09.006

Figure Lengend Snippet: Functional Knockout of Endogenous CXCR4 in Human Jurkat Cells (A) Scheme of the functional CXCR4 knockout experiment. Human Jurkat cells sorted for high CD4 and CXCR4 expression were transduced with LIT or Gag.MS2 CRISPR/Cas9 all-in-one particles for targeted CXCR4 knockout. In a second round of transduction, CXCR4 knockout cultures were transduced with a CXCR4-tropic LIT.EGFP vector. Only cells that still carry the CXCR4 receptor will be susceptible to transduction with CXCR4-tropic LIT.EGFP. The flow cytometric plots show CD4 and CXCR4 expression in Jurkat cells before and after sorting. (B) Schemes of LIT.CRISPR/Cas9.CXCR4 all-in-one and LIT.EGFP vectors. Depicted is the vector configuration after integration. All vectors are self-inactivating (SIN) due to deletion of viral enhancer and promoter sequences within the long terminal repeats (LTRs), and transgene expression ( SpCas9.P2A.dTomato or EGFP ) is driven by an internal spleen focus-forming virus (SFFV) promoter. In the LIT.CRISPR/Cas9.CXCR4 all-in-one vector, the hU6 promoter drives expression of sgRNA.CXCR4. Ψ, packaging signal; RRE, rev responsive element; cPPT, central polypurine tract; PRE, post-transcriptional regulatory element from woodchuck hepatitis virus. (C) Efficient CXCR4 knockout by Gag.MS2.CRISPR/Cas9 all-in-one particles. Sorted CD4 + /CXCR4 + Jurkat cells were transduced with LIT.CRISPR/Cas9.CXCR4 all-in-one particles (MOI 5) or either 25 μL of 100-fold concentrated (CXCR4.TS.inc and CXCR4.TS.adj) or 50 μL of 50-fold concentrated (Tp53.TS.inc and Tp53.TS.adj) Gag.MS2.CRISPR/Cas9 all-in-one particles. The knockout rate was determined by flow cytometry 5 days after transduction. Each data point reflects one independently generated supernatant. (D) Genome targeting efficiencies of LIT.CRISPR/Cas9.CXCR4 and Gag.MS2.CRISPR/Cas9.CXCR4.TS.adj all-in-one particles. The gDNA of CXCR4 knockout cultures was subjected to a T7 endonuclease I assay. On-target activity is indicated by the cleavage of the 940 bp CXCR4 PCR amplicon into 580 and 360 bp fragments. The InDel rate of each sample is depicted as a percentage. (E) Reduced CXCR4-tropic LIT.EGFP transduction rates in cultures that were treated with Gag.MS2.CRISPR/Cas9.CXCR4.TS.adj all-in-one particles. LIT.CRISPR/Cas9.CXCR4 all-in-one and Gag.MS2.CRISPR/Cas9.CXCR4.TS.adj cultures from (C) were individually transduced with CXCR4-tropic LIT.EGFP vector particles (9 days after CRISPR/Cas9 treatment). Three days later, cultures were analyzed by flow cytometry for EGFP and CXCR4 expression. The right graph depicts the ratios EGFP + /EGFP − cells in CXCR4 + or CXCR4 − fractions expressed as mean ± SD. Representative flow cytometry plots are shown on the left (MFIs in red).

Article Snippet: After staining all blotted proteins with Ponceau S (Sigma-Aldrich), the membrane was successively probed with a rabbit polyclonal anti-enterobacteriophage MS2 coat protein antibody (1:5,000) (Merck Millipore, Darmstadt, Germany) and a monoclonal mouse purified anti-CRISPR (Cas9) antibody (1:1,500) (BioLegend, Koblenz, Germany) in Tris-buffered saline with 0.05% Tween and 3% milk powder at 4°C overnight (both Roth, Karlsruhe, Germany).

Techniques: Functional Assay, Knock-Out, Expressing, Transduction, CRISPR, Plasmid Preparation, Virus, Flow Cytometry, Generated, T7EI Assay, Activity Assay, Amplification

Journal: Molecular Therapy. Nucleic Acids

Article Title: Transient Retrovirus-Based CRISPR/Cas9 All-in-One Particles for Efficient, Targeted Gene Knockout

doi: 10.1016/j.omtn.2018.09.006

Figure Lengend Snippet: Growth Advantage of NUFF TP53 Knockout Cells (A) Generation of DsRedexp + NUFF cells. Freshly thawed NUFF cells (passage 9) were stably transduced with LIT.DsRedexp.IRES.Zeo encoding for DsRedexp and the Zeocin resistance gene (Zeo) at MOI 0.5. Co-expression of DsRedexp and Zeo was achieved by the internal ribosomal entry site (IRES) of endomycarditis virus and was initiated by enhancer and promoter sequences of SFFV. Transduced NUFF cells were selected with 100 μg/mL Zeocin for 10 days. The flow cytometric plots depict the percentage of DsRedexp + NUFF cells before and after Zeocin selection. (B) Knockout of TP53 , but not PTEN , resulted in a growth advantage. DsRedexp + NUFF cells were transduced in a single round with LIT.CRISPR/Cas9 all-in-one vector particles for targeted knockout of PTEN , TP53 , or Luc at MOI 10. Cells were mixed with non-treated wild-type NUFF cells 3 days post-transduction, and co-cultures were analyzed for DsRedexp expression for up to 67 days. The control co-culture of non-treated DsRedexp + and wild-type NUFF cells is marked in red. Exp 1, experiment 1; Exp 2, experiment 2. (C) Outgrowth of DsRedexp + NUFF cells that were treated with Gag.MS2.CRISPR/Cas9.TP53 all-in-one particles. DsRedexp + NUFF cells were transduced once with 50 μL of the depicted non-integrating Gag.MS2.CRISPR/Cas9 all-in-one particles. In TS.inc and TS.adj sgRNA combined samples, the respective supernatants were mixed at a ratio of 1:1 before transduction. (D) Transduction of DsRedexp + NUFF cells twice with Gag.MS2.CRISPR/Cas9.TP53 all-in-one particles slightly accelerates outgrowth of TP53 knockout cells. The experiment was similar to that in (C), but DsRedexp + NUFF cells were transduced twice with Gag.MS2.CRISPR/Cas9 all-in-one particles on 2 consecutive days before co-culture. (E) Double knockout of PTEN and TP53 by Gag.MS2.CRISPR/Cas9 all-in-one particles did not accelerate outgrowth of DsRedexp + NUFF cells. Before co-culture, DsRedexp + NUFF cells were co-transduced with either LIT or Gag.MS2 CRISPR/Cas9 all-in-one particles targeting TP53 or PTEN . LIT.CRISPR/Cas9 all-in-one supernatants were each applied at MOI 10, and 50 μL of each Gag.MS2.CRISPR/Cas9 all-in-one supernatant was used. The curves were obtained from individual transductions with independently generated supernatants. Numbers 1–9 (in blue) displayed in graphs (B)–(E) indicate selected cultures that were expanded for gDNA harvest and subjected to InDel analyses (see also ).

Article Snippet: After staining all blotted proteins with Ponceau S (Sigma-Aldrich), the membrane was successively probed with a rabbit polyclonal anti-enterobacteriophage MS2 coat protein antibody (1:5,000) (Merck Millipore, Darmstadt, Germany) and a monoclonal mouse purified anti-CRISPR (Cas9) antibody (1:1,500) (BioLegend, Koblenz, Germany) in Tris-buffered saline with 0.05% Tween and 3% milk powder at 4°C overnight (both Roth, Karlsruhe, Germany).

Techniques: Knock-Out, Stable Transfection, Transduction, Expressing, Virus, Selection, CRISPR, Plasmid Preparation, Control, Co-Culture Assay, Double Knockout, Generated