cd63  (OriGene)

Journal: Nature Communications

Article Title: A modular strategy for extracellular vesicle-mediated CRISPR-Cas9 delivery through aptamer-based loading and UV-activated cargo release

doi: 10.1038/s41467-025-65995-3

Figure Lengend Snippet: A Schematic of the EV engineering strategy for active loading and delivery of Cas9 RNPs in HEK293T-derived EVs. Tandem MCPs, lacking the Fg loop involved in capsid formation, are intraluminally fused to the N-terminus of EV-enriched CD63 (MCP-CD63). These MCPs bind MS2 aptamers protruding from the RNP, which are present on MS2-modified sgRNAs. Created in BioRender. Utrecht University, P. (2025) https://BioRender.com/0up6d3r . B Nanosight particle tracking analysis displaying the size distribution of isolated EVs. Means ± SD, n = 5 technical replicates. Representative NTA analysis, observed > 10 times in independent experiments. C Transmission electron microscopy image of isolated EVs, scalebar represents 200 nm. Representative TEM analysis, observed >3 times in independent experiments. D Western blot analysis of cell lysates and EVs shows an enrichment for EV markers CD63, ALIX and TSG101, and a negative enrichment for the ER organelle marker Calnexin, in isolated EVs. Due to the highly glycosylated nature of CD63, its western blot analysis presents a commonly observed “smear” pattern. Representative Western Blots, observed > 3 times in independent experiments. E Western blot analysis shows similar levels of Cas9 in cell lysates regardless of co-expression of MCP-CD63, whereas high levels of Cas9 in isolated EVs are only observed upon co-expression of MCP-CD63. Representative Western Blots, observed 2 times in independent experiments. F Western blot analysis of an OptiPrep density gradient of isolated EVs shows presence of Cas9 in the same EV-associated fractions as EV-marker CD63, n = 1. G , H qPCR analysis ( G ) of RNA isolated from EVs derived from cells MS2-sgRNA with- or without the co-expression of MCP-CD63 shows enriched loading of MS2-sgRNAs into isolated EVs, which is further quantified by ddPCR analysis ( H ), corrected for NTA particle count and Spike-in RNA, to calculate RNA isolation efficiency, into absolute EV per sgRNA counts. Means + SD, n = 3 biologically independent samples, Student’s t test. * p < 0.05, *** p < 0.001.

Article Snippet: Membranes were then probed overnight at 4 o C in staining buffer consisting of 1 part Intercept Blocking Buffer (LI-COR Biosciences) and 1 part Tris-Buffered Saline with 0.1% Tween-20 (TBS-T), using the following primary antibodies: ALIX 1:1000 (Thermo Fisher Scientific, MA1-83977), Calnexin 1:1000 (GeneTex, GTX101676), CD63 1:1000 (AB8219), Syntenin 1:500 (Origene, OTI2H6), TSG101 1:1000 (Abcam, ab30871), and H2B 1:1000 (Abcam, ab52599), GAPDH 1:500 (Abcam, AB9485) or α-Flag 1:1000 (Sigma-Aldrich, F1804).

Techniques: Derivative Assay, Modification, Isolation, Transmission Assay, Electron Microscopy, Western Blot, Marker, Expressing

Journal: Nature Communications

Article Title: A modular strategy for extracellular vesicle-mediated CRISPR-Cas9 delivery through aptamer-based loading and UV-activated cargo release

doi: 10.1038/s41467-025-65995-3

Figure Lengend Snippet: A Schematic of the fluorescence “stoplight” reporter construct for Cas9 activity. mCherry (red) is stably expressed under a CMV promoter followed by a small “linker” region (blue), containing a Cas9 target site, and a stop codon. Frameshifts in the linker region, resulting from non-homologous end joining (NHEJ)-mediated repair mechanisms due to Cas9-mediated double stranded breaks, result in expression of downstream eGFP (green) open reading frames. Created in BioRender. Utrecht University, P. (2025) https://BioRender.com/olgq16g . B , C Fluorescence microscopy images ( B ) and flow cytometry analysis ( C ) of HEK293T cells expressing the stoplight reporter construct, transfected with Cas9 and a non-targeting (NT) sgRNA, or a targeting (T) sgRNA with or without MS2 aptamers. eGFP expression is observed after transfection of Cas9 with both wild-type targeting (T) sgRNA or a T MS2-sgRNA. Scalebar represents 200 μm. Means + SD, n = 3 biologically independent samples, One-way ANOVA with post-hoc Tukey’s multiple comparisons test. D , E Flow cytometry analysis ( D ) and fluorescence microscopy images ( E ) of HEK293T cells expressing the stoplight reporter construct, 72 h after addition of EVs isolated from HEK293T cells expressing Cas9 + MS2-sgRNA + VSV-G (EV RNP) or expressing Cas9 + MS2-sgRNA + MCP-CD63 + VSV-G (EV RNP + MCP-CD63) shows that MCP-CD63 facilitates a significant but limited increase of EV-mediated RNP delivery. 1.0 × 10 12 EVs per well. Scalebar represents 200 μm. Means + SD, n = 3 biologically independent samples, One-way ANOVA with post-hoc Tukey’s multiple comparisons test. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Membranes were then probed overnight at 4 o C in staining buffer consisting of 1 part Intercept Blocking Buffer (LI-COR Biosciences) and 1 part Tris-Buffered Saline with 0.1% Tween-20 (TBS-T), using the following primary antibodies: ALIX 1:1000 (Thermo Fisher Scientific, MA1-83977), Calnexin 1:1000 (GeneTex, GTX101676), CD63 1:1000 (AB8219), Syntenin 1:500 (Origene, OTI2H6), TSG101 1:1000 (Abcam, ab30871), and H2B 1:1000 (Abcam, ab52599), GAPDH 1:500 (Abcam, AB9485) or α-Flag 1:1000 (Sigma-Aldrich, F1804).

Techniques: Fluorescence, Construct, Activity Assay, Stable Transfection, Non-Homologous End Joining, Expressing, Microscopy, Flow Cytometry, Transfection, Isolation

Journal: Nature Communications

Article Title: A modular strategy for extracellular vesicle-mediated CRISPR-Cas9 delivery through aptamer-based loading and UV-activated cargo release

doi: 10.1038/s41467-025-65995-3

Figure Lengend Snippet: A Schematic of the EV engineering strategy for photo-activatable release of membrane-bound MCP-loaded Cas9 RNPs. A photocleavable domain (PhoCl), with an N-terminal HA-tag for western blot analysis, is placed between the tandem MCPs and CD63 (MCP-PhoCl-CD63). Upon exposure to 395 nm UV light, PhoCl is cleaved, releasing the MCP-RNP complex from the EV membrane. Created in BioRender. Utrecht University, P. (2025) https://BioRender.com/r3y3yik . B , C Western blot analysis of MCP-PhoCl-CD63 cleavage in cells ( B ) and isolated EVs ( C ). Upon UV exposure, the ~ 82 kDa fusion protein is cleaved, revealing a ~ 55 kDa cleavage product. D , E Flow cytometry analysis ( D ) and fluorescence microscopy images ( E ) of HEK293T cells expressing the stoplight reporter construct, 72 h after addition of EVs isolated from HEK293T cells expressing Cas9 + MS2-sgRNA + MCP-PhoCl-CD63 + VSV-G shows that UV treatment of EVs prior to addition to cells strongly increases EV-mediated RNP delivery. 1.0 × 10 12 EVs per well. Mean + SD, n = 5 independent experiments, One-way ANOVA with post-hoc Tukey’s multiple comparisons test. Scalebar represents 200 μm. F Schematic of additional EV-targeted loading constructs. Tandem MCPs are fused to EV-enriched moieties CD9, CD63, CD81, ARRDC1, and a myristoylation tag via a photocleavable (PhoCl) domain. Created in BioRender. Utrecht University, P. (2025) https://BioRender.com/f95r962 . G , H Flow cytometry analysis ( G ) and fluorescence microscopy images ( H ) comparing EV-mediated RNP delivery of various MCP-PhoCl fusion proteins. Addition normalized by particle count; 5.0 × 10 10 particles added per well. Scalebar represents 200 μm. Means + SD, n = 3 independent experiments, One-way ANOVA with post-hoc Tukey’s multiple comparisons test. I Western blot analysis of Cas9 loading in EVs by various MCP-PhoCl fusion proteins. Relative Cas9 loading is corrected for loading control and normalized to the “RNP only” condition. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: Membranes were then probed overnight at 4 o C in staining buffer consisting of 1 part Intercept Blocking Buffer (LI-COR Biosciences) and 1 part Tris-Buffered Saline with 0.1% Tween-20 (TBS-T), using the following primary antibodies: ALIX 1:1000 (Thermo Fisher Scientific, MA1-83977), Calnexin 1:1000 (GeneTex, GTX101676), CD63 1:1000 (AB8219), Syntenin 1:500 (Origene, OTI2H6), TSG101 1:1000 (Abcam, ab30871), and H2B 1:1000 (Abcam, ab52599), GAPDH 1:500 (Abcam, AB9485) or α-Flag 1:1000 (Sigma-Aldrich, F1804).

Techniques: Membrane, Western Blot, Isolation, Flow Cytometry, Fluorescence, Microscopy, Expressing, Construct, Control

Journal: Nature Communications

Article Title: A modular strategy for extracellular vesicle-mediated CRISPR-Cas9 delivery through aptamer-based loading and UV-activated cargo release

doi: 10.1038/s41467-025-65995-3

Figure Lengend Snippet: A Schematic of the fluorescent reporter construct for transcriptional activation; pInducer20-eGFP. An eGFP open reading frame is placed after a Tet Responsive Element (TRE) sequence. Transcription of eGFP can either be facilitated by activation of the co-expressed reverse tet-transactivator rTA3 by addition of doxycycline (1) or by introduction of transcriptional activator dCas9-VPR with a sgRNA targeting the TRE sequence (2). Created in BioRender. Utrecht University, P. (2025) https://BioRender.com/v9au9y6 . B , C Fluorescence microscopy images ( B ) and flow cytometry analysis ( C ) of HEK293T cells expressing the pInducer20-eGFP reporter construct, 48 h after addition of doxycycline (0.5 µg/ml), or transfection with plasmids encoding for dCas9-VPR with non-targeting (NT) sgRNAs, targeting (T) sgRNAs, or targeting MS2-sgRNAs. Doxycycline and dCas9-VPR with targeting sgRNAs increase eGFP expression. MFI: mean fluorescence intensity. Scalebar represents 200 μm. Means + SD, n = 3 biologically independent samples, One-way ANOVA with post-hoc Tukey’s multiple comparisons test. D Flow cytometry analysis of HEK293T cells expressing the pInducer20-eGFP reporter construct, 48 h after addition of EVs from HEK293T expressing dCas9-VPR alongside either MCP-PhoCl-CD63 or MCP-PhoCl-CD9, in combination with non-targeting (NT), or targeting (T) sgRNAs. Both loading constructs facilitate EV-mediated functional dCas9-VPR delivery, resulting in a significant increase in eGFP mean fluorescence intensity (MFI). 4.0 × 10 11 EVs per well. Means + SD, n = 5 independent experiments, One-way ANOVA with post-hoc Dunnett’s multiple comparisons test. * p < 0.05, *** p < 0.001, **** p < 0.0001.

Article Snippet: Membranes were then probed overnight at 4 o C in staining buffer consisting of 1 part Intercept Blocking Buffer (LI-COR Biosciences) and 1 part Tris-Buffered Saline with 0.1% Tween-20 (TBS-T), using the following primary antibodies: ALIX 1:1000 (Thermo Fisher Scientific, MA1-83977), Calnexin 1:1000 (GeneTex, GTX101676), CD63 1:1000 (AB8219), Syntenin 1:500 (Origene, OTI2H6), TSG101 1:1000 (Abcam, ab30871), and H2B 1:1000 (Abcam, ab52599), GAPDH 1:500 (Abcam, AB9485) or α-Flag 1:1000 (Sigma-Aldrich, F1804).

Techniques: Construct, Activation Assay, Sequencing, Fluorescence, Microscopy, Flow Cytometry, Expressing, Transfection, Functional Assay

Journal: Nature Communications

Article Title: A modular strategy for extracellular vesicle-mediated CRISPR-Cas9 delivery through aptamer-based loading and UV-activated cargo release

doi: 10.1038/s41467-025-65995-3

Figure Lengend Snippet: A Schematic of the fluorescent reporter construct for ABE activity. mCherry is followed by a stop codon and an in-frame eGFP ORF. A Cas9 target site is present at the mCherry stop codon, allowing ABE-mediated conversion of the stop codon into glutamine (Gln), resulting in the expression of a mCherry-eGFP fusion protein. Created in BioRender. Utrecht University, P. (2025) https://BioRender.com/90akg9p . B EV-mediated delivery of ABE8e using MCP-PhoCl-CD63 or MCP-PhoCl-CD9 with a targeting MS2-sgRNA 2.0 results in significant but limited ABE8e activity. 5.0 × 10 10 EVs per well. Means + SD, n = 3 independent experiments, One-way ANOVA with post-hoc Tukey’s multiple comparisons test. C , D Transfection of plasmid DNA encoding for ABE8e with a targeting WT sgRNA (no MS2 stemloops), MS2-sgRNA 1.1 (MS2 aptamer in tetraloop), MS2-sgRNA 1.2 (MS2 aptamers in stem loop 2), or MS2-sgRNA 2.0 (MS2 aptamer in tetraloop and stemloop 2) results in similar levels of ABE8e activity, as shown by fluorescence microscopy images ( C ) and flow cytometry analysis ( D ), 48 h after transfection. Scalebar represents 200 μm. Means + SD, n = 3 biologically independent samples. E Flow cytometry analysis of HEK293T fluorescent reporter cells transfected with ABE8e and various targeting sgRNAs, alongside a plasmid for expression of cytosolic tandem MCPs, or an empty control vector. Only sgRNAs with an MS2 aptamer in the second stemloop show decreased ABE8e activity in the presence of free MCPs. Means + SD, n = 3 biologically independent samples, One-way ANOVA with post-hoc Sidak’s multiple comparisons test. F , G , H Flow cytometry ( F , G ) and fluorescence microscopy analysis ( H ) for a dose response of EV-mediated ABE8e delivery using MCP-PhoCl-CD63 ( F ) and MCP-PhoCl-CD9 ( G ) comparing MS2-sgRNA 1.1 and 2.0 after addition of 1.0 × 10 10 , 5.0 × 10 10 , and 1.0 × 10 11 EVs. Both loading constructs show high ABE8e activity with MS2-sgRNA 1.1, in a dose-dependent manner. Scalebar represents 200 μm. Means + SD, n = 3 independent experiments, One-way ANOVA with post-hoc Sidak’s multiple comparisons test. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: Membranes were then probed overnight at 4 o C in staining buffer consisting of 1 part Intercept Blocking Buffer (LI-COR Biosciences) and 1 part Tris-Buffered Saline with 0.1% Tween-20 (TBS-T), using the following primary antibodies: ALIX 1:1000 (Thermo Fisher Scientific, MA1-83977), Calnexin 1:1000 (GeneTex, GTX101676), CD63 1:1000 (AB8219), Syntenin 1:500 (Origene, OTI2H6), TSG101 1:1000 (Abcam, ab30871), and H2B 1:1000 (Abcam, ab52599), GAPDH 1:500 (Abcam, AB9485) or α-Flag 1:1000 (Sigma-Aldrich, F1804).

Techniques: Construct, Activity Assay, Expressing, Transfection, Plasmid Preparation, Fluorescence, Microscopy, Flow Cytometry, Control