Journal: Journal of Extracellular Vesicles

Article Title: A platform for actively loading cargo RNA to elucidate limiting steps in EV-mediated delivery

doi: 10.3402/jev.v5.31027

Figure Lengend Snippet: Evaluation of RNA loading into EVs via the TAMEL platform. (a) This cartoon summarizes the concept of facilitating active loading of cargo RNA into EVs via our TAMEL platform. A TAMEL EV-loading protein comprises an EV-enriched protein (EEP, blue) fused to an RNA-binding domain (RBD, green), which localizes to EVs. Actively loaded RNA (green) contains a motif that binds to the RBD, resulting in enhanced loading into EVs relative to passively loaded RNA (orange). (b) RNA cargo design impacts active loading. The “fold enrichment of cargo mRNA +/−MS2” is defined as the ratio of cargo RNA/GAPDH mRNA in EVs derived from cells expressing Lamp2b–MS2–HA divided by the same RNA ratio in EVs derived from cells expressing Lamp2b–HA. All experiments were performed in biological triplicates. (c) Cartoon illustrating the 3′ RNA fragment analysis technique. Cargo RNA is first reverse transcribed using an oligo dT primer, and amplicons corresponding to the RNA 5′ or 3′ ends (the latter is located ~500 bases upstream of the polyA site) are then quantified by qPCR using the primer pairs indicated. Note that the amplicon near the RNA 3′ end will be present in cDNA derived from both full-length RNA and 3′ RNA fragments. (d) Analysis of 3′ RNA fragment loading into EVs. Cargo RNA levels were quantified as depicted in panel c and normalized to GAPDH. Passive loading: cells transfected with Lamp2b–HA; active loading: cells transfected with Lamp2b–MS2–HA. (e) Full-length RNA and 3′ fragment RNA levels in EVs were quantified following incubation at 37°C; experiments were performed in technical duplicate with a biological replicate shown in Supplementary Fig. 2a. Error bars indicate 1 standard deviation, throughout. MVB, multivesicular body.

Article Snippet: The pMS2-GFP plasmid, which encodes the MS2 bacteriophage coat protein tandem dimer (mutant d1FG and V29I ( , )), was obtained from Addgene (plasmid #27121), deposited by Robert Singer ( ).

Techniques: RNA Binding Assay, Derivative Assay, Expressing, Reverse Transcription, Amplification, Transfection, Incubation, Standard Deviation

Journal: Journal of Extracellular Vesicles

Article Title: A platform for actively loading cargo RNA to elucidate limiting steps in EV-mediated delivery

doi: 10.3402/jev.v5.31027

Figure Lengend Snippet: Impact of EEP choice on TAMEL-mediated active RNA loading into vesicles. (a) Effects of EEP choice on cargo RNA loading into EVs. Experiments were performed in biological triplicate. (b) Effects of EEP choice on cargo loading into gesicles. Experiments were performed in biological triplicate. (c) Protein abundance was quantified by densitometry analysis of anti-HA western blots (Supplementary Fig. 3), and each blot was internally normalized by the intensity for VSVG–MS2–HA in gesicles (maximal intensity case). This experiment was performed in biological duplicate. The y-axis is in log scale to enable visualization of all values. Error bars indicate 1 standard deviation, throughout.

Article Snippet: The pMS2-GFP plasmid, which encodes the MS2 bacteriophage coat protein tandem dimer (mutant d1FG and V29I ( , )), was obtained from Addgene (plasmid #27121), deposited by Robert Singer ( ).

Techniques: Quantitative Proteomics, Western Blot, Standard Deviation

Journal: Journal of Extracellular Vesicles

Article Title: A platform for actively loading cargo RNA to elucidate limiting steps in EV-mediated delivery

doi: 10.3402/jev.v5.31027

Figure Lengend Snippet: Comparative analysis of dTomato delivery by actively or passively loaded vesicles. For panels a–c, the cartoons at left summarize the experimental designs, and in the panels at right, each data point represents the average of duplicate wells of cells treated with the same type of vesicle. Error bars indicate 1 standard deviation. “Normalized fluorescence” is defined as the mean fluorescence of cells receiving vesicles divided by the mean fluorescence of cells receiving a medium change only. (a) Time course of EV delivery to cells. Grey squares: CD63–HA EVs; orange circles: CD63–MS2–HA EVs. The solid arrow represents cells that received a medium change after 4 h of EV treatment. The dashed arrow indicates that cells did not receive a medium change. Statistically significant differences (p<0.05, not shown for clarity): CD63–MS2–HA +/− medium change. An independent repeat of this experiment is shown in Supplementary Fig. 3a. (b) Time course of gesicle delivery to cells. Purple squares: VSVG–HA gesicles; green circles: VSVG–MS2–HA gesicles. Solid and dashed arrows carry the same meaning as in panel (a). Statistically significant differences (p < 0.05, not shown for clarity): VSVG–HA versus VSVG–MS2–HA at 4 and 16 h (comparisons were made for each time point), VSVG–HA +/− medium change, and VSVG–MS2–HA +/− medium change. An independent repeat of this experiment is shown in Supplementary Fig. 3c. (c) Comparison of delivery by gesicles from cells transfected with VSVG–HA (purple), VSVG–MS2–HA (green) or a 50:50 mix of VSVG–HA and VSVG–MS2–HA (hybrid gesicles, magenta). (d) dTomato RNA levels (normalized to GAPDH) in VSVG–HA gesicles (purple), VSVG–MS2–HA gesicles (green) or hybrid gesicles (magenta). Error bars indicate 1 standard deviation of technical duplicate samples. *Significant difference was evaluated with a Student's t -test using a cut-off of p < 0.05.

Article Snippet: The pMS2-GFP plasmid, which encodes the MS2 bacteriophage coat protein tandem dimer (mutant d1FG and V29I ( , )), was obtained from Addgene (plasmid #27121), deposited by Robert Singer ( ).

Techniques: Standard Deviation, Fluorescence, Comparison, Transfection

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: 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