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Cited by 117CRISPR and Genetic EngineeringRNA Interference and Gene DeliveryVirus-based gene therapy research

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VSV-G-Enveloped Vesicles for Traceless Delivery of CRISPR-Cas9

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Montagna, Claudia, Petris, Gianluca, Casini, Antonio, Maule, Giulia, Franceschini, Gian Marco, Zanella, Ilaria, Conti, Luciano, Arnoldi, Francesca, Burrone, Oscar R., Zentilin, Lorena, Zacchigna, Serena, Giacca, Mauro, Cereseto, Anna·2018·Molecular Therapy Nucleic Acids
DOI 10.1016/j.omtn.2018.05.010PMC PMC6041463
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Abstract

The method of delivery of CRISPR-Cas9 into target cells is a strong determinant of efficacy and specificity in genome editing. Even though high efficiency of Cas9 delivery is necessary for optimal editing, its long-term and high levels of expression correlate with increased off-target activity. We developed vesicles (VEsiCas) carrying CRISPR-SpCas9 ribonucleoprotein complexes (RNPs) that are efficiently delivered into target cells through the fusogenic glycoprotein of the vesicular stomatitis virus (VSV-G). A crucial step for VEsiCas production is the synthesis of the single guide RNA (sgRNA) mediated by the T7 RNA polymerase in the cytoplasm of producing cells as opposed to canonical U6-driven Pol III nuclear transcription. In VEsiCas, the absence of DNA encoding SpCas9 and sgRNA allows rapid clearance of the nuclease components in target cells, which correlates with reduced genome-wide off-target cleavages. Compared with SpCas9 RNPs electroporation, which is currently the method of choice to obtain transient SpCas9 activity, VEsiCas deliver the nuclease with higher efficiency and lower toxicity. We show that a wide variety of cells can be edited through VEsiCas, including a variety of transformed cells, induced pluripotent stem cells (iPSCs), and cardiomyocytes, in vivo. VEsiCas is a traceless CRISPR-Cas9 delivery tool for efficient and safe genome editing that represents a further advancement toward the therapeutic use of the CRISPR-Cas9 technology.

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Figure 1 Design and Genome Editing Activity of VEsiCas (A) EGFP disruption assay with VSV-G/SpCas9 vesicles produced in HEK293T cells. Shown are percentages of EGFP knockout HEK293-EGFP cells generated by transfection of SpCas9 (SpCas9 plasmid) together with EGFP targeting (sgEGFP5) or control (sgCtr) sgRNA or transduction with VSV-G/SpCas9 vesicles carrying U6-transcribed sgRNA. Where indicated, HEK293-EGFP cells were pre-transfected with sgEGFP5 or sgCtr (+ pre-sgRNA) prior to VSV-G/SpCas9 vesicle treatment. Data are presented as mean ± SEM for n = 2 independent experiments. (B) Schematic of VEsiCas production in BSR-T7/5 cells. T7 RNA polymerase, expressed in the cytosol, regulates cytosolic sgRNA expression by means of the T7 promoter. Vesicles decorated with VSV-G, expressed by BSR-T7/5 producer cells, bud incorporating SpCas9 complexed with sgRNA to form VEsiCas. In target cells, VEsiCas release active SpCas9-sgRNA complexes that enter the nuclei through two nuclear localization sequences introduced in SpCas9. (C) Genome activity of VEsiCas produced in BSR-T7/5 on HEK293-EGFP cells. Shown are percentages of non-fluorescent HEK293-EGFP cells following transfection of SpCas9 (SpCas9 plasmid) together with sgRNAs (sgEGFP5 or sgCtr) or treatment with VEsiCas carrying sgRNAs (sgEGFP5 or sgCtr) either with or without pre-transfection with sgRNAs, as indicated. Data are presented as mean ± SEM for n = 2 independent experiments. (D and E) VEsiCas-mediated editing of the CXCR4 (D) and VEGFA site3 (E) genomic loci. Percentages of indel formation in HEK293T cells were measured through TIDE analysis following transfection of SpCas9 (SpCas9 plasmid) together with sgRNAs (sgCXCR4, sgVEGFA site3, or sgCtr) or after three sequential treatments with VEsiCas carrying sgRNAs (sgCXCR4, sgVEGFA site3, or sgCtr). Data are presented as mean ± SEM for n = 2 independent experiments.
Figure 1 Design and Genome Editing Activity of VEsiCas (A) EGFP disruption assay with VSV-G/SpCas9 vesicles produced in HEK293T cells. Shown are percentages of EGFP knockout HEK293-EGFP cells generated by transfection of SpCas9 (SpCas9 plasmid) together with EGFP targeting (sgEGFP5) or control (sgCtr) sgRNA or transduction with VSV-G/SpCas9 vesicles carrying U6-transcribed sgRNA. Where indicated, HEK293-EGFP cells were pre-transfected with sgEGFP5 or sgCtr (+ pre-sgRNA) prior to VSV-G/SpCas9 vesicle treatment. Data are presented as mean ± SEM for n = 2 independent experiments. (B) Schematic of VEsiCas production in BSR-T7/5 cells. T7 RNA polymerase, expressed in the cytosol, regulates cytosolic sgRNA expression by means of the T7 promoter. Vesicles decorated with VSV-G, expressed by BSR-T7/5 producer cells, bud incorporating SpCas9 complexed with sgRNA to form VEsiCas. In target cells, VEsiCas release active SpCas9-sgRNA complexes that enter the nuclei through two nuclear localization sequences introduced in SpCas9. (C) Genome activity of VEsiCas produced in BSR-T7/5 on HEK293-EGFP cells. Shown are percentages of non-fluorescent HEK293-EGFP cells following transfection of SpCas9 (SpCas9 plasmid) together with sgRNAs (sgEGFP5 or sgCtr) or treatment with VEsiCas carrying sgRNAs (sgEGFP5 or sgCtr) either with or without pre-transfection with sgRNAs, as indicated. Data are presented as mean ± SEM for n = 2 independent experiments. (D and E) VEsiCas-mediated editing of the CXCR4 (D) and VEGFA site3 (E) genomic loci. Percentages of indel formation in HEK293T cells were measured through TIDE analysis following transfection of SpCas9 (SpCas9 plasmid) together with sgRNAs (sgCXCR4, sgVEGFA site3, or sgCtr) or after three sequential treatments with VEsiCas carrying sgRNAs (sgCXCR4, sgVEGFA site3, or sgCtr). Data are presented as mean ± SEM for n = 2 independent experiments.CC BY-NC-ND · PubMed Central
Figure 2 Genome Editing by Multiplexed VEsiCas (A) Gene deletion using VEsiCas. Shown is gel electrophoresis analysis of the EGFP locus deletion obtained in HEK293-EGFP cells through Multi-VEsiCas (right) treatment or by transfection with SpCas9 together with specific sgRNAs (sgEGFP5 and sgEGFPBi) (left). The amount of deletion (percent) was quantified by densitometry. Arrowheads indicate the expected band corresponding to PCR amplification of the deleted EGFP locus. (B) Activity of VEsiCas delivering SpCas9 nickase. Shown are percentages of EGFP knockout cells following incubation with VEsiCas-n, carrying SpCas9 nickase loaded with sgRNA toward two sites in the EGFP locus (sgEGFP3gW and sgEGFPBi) or with sgCtr. NT, not treated. Data are presented as mean ± SEM for n = 2 independent experiments.
Figure 2 Genome Editing by Multiplexed VEsiCas (A) Gene deletion using VEsiCas. Shown is gel electrophoresis analysis of the EGFP locus deletion obtained in HEK293-EGFP cells through Multi-VEsiCas (right) treatment or by transfection with SpCas9 together with specific sgRNAs (sgEGFP5 and sgEGFPBi) (left). The amount of deletion (percent) was quantified by densitometry. Arrowheads indicate the expected band corresponding to PCR amplification of the deleted EGFP locus. (B) Activity of VEsiCas delivering SpCas9 nickase. Shown are percentages of EGFP knockout cells following incubation with VEsiCas-n, carrying SpCas9 nickase loaded with sgRNA toward two sites in the EGFP locus (sgEGFP3gW and sgEGFPBi) or with sgCtr. NT, not treated. Data are presented as mean ± SEM for n = 2 independent experiments.CC BY-NC-ND · PubMed Central
Figure 3 Genome Editing Activity in Human iPSCs and in the Heart of an EGFP Mouse Model (A) VEsiCas activity in induced pluripotent stem cells (iPSCs). Shown is the percentage of non-fluorescent iPSCs stably expressing EGFP after treatment with VEsiCas carrying either sgCtr or sgGFPI2. Data are presented as mean ± SEM for n = 2 independent experiments. (B) VEsiCas-mediated gene disruption in the cardiac muscle of EGFP mice. Shown are fluorescence microscopy images of cardiac tissue sections from EGFP transgenic mice 10 days after intra-cardiac injection of VEsiCas carrying sgCtr or sgEGFPBi. DAPI was used as a nuclear counterstain. Immunostaining for α-actinin was performed to identify cardiomyocytes (bottom). A representative sample of n = 5 experiments is shown. Scale bar, 100 μm. (C) Quantification of VEsiCas EGFP knockout in cardiac tissue. The bar graph shows the percentage of non-fluorescent cardiomyocytes following VEsiCas treatments (mean ± SEM of n = 5 independent experiments).
Figure 3 Genome Editing Activity in Human iPSCs and in the Heart of an EGFP Mouse Model (A) VEsiCas activity in induced pluripotent stem cells (iPSCs). Shown is the percentage of non-fluorescent iPSCs stably expressing EGFP after treatment with VEsiCas carrying either sgCtr or sgGFPI2. Data are presented as mean ± SEM for n = 2 independent experiments. (B) VEsiCas-mediated gene disruption in the cardiac muscle of EGFP mice. Shown are fluorescence microscopy images of cardiac tissue sections from EGFP transgenic mice 10 days after intra-cardiac injection of VEsiCas carrying sgCtr or sgEGFPBi. DAPI was used as a nuclear counterstain. Immunostaining for α-actinin was performed to identify cardiomyocytes (bottom). A representative sample of n = 5 experiments is shown. Scale bar, 100 μm. (C) Quantification of VEsiCas EGFP knockout in cardiac tissue. The bar graph shows the percentage of non-fluorescent cardiomyocytes following VEsiCas treatments (mean ± SEM of n = 5 independent experiments).CC BY-NC-ND · PubMed Central
Figure 4 Analysis of On/Off-Target Activity Generated by VEsiCas on the VEGFA Locus (A) Traceless delivery of SpCas9 through VEsiCas. Shown is a time course analysis of SpCas9 intracellular levels by western blot analysis in HEK293T cells transduced with VEsiCas or transfected with a plasmid expressing SpCas9. The reported time points correspond to the time of analysis following treatments (transduction or transfection). The western blot is representative of n = 2 independent experiments. (B) Targeted comparison of genome editing specificity using VEsiCas or a plasmid expressing SpCas9 in combination with sgVEGFA site3. Shown are percentages of indel formation measured by TIDE in the VEGFA site3 locus and in two sgVEGFA site3 previously validated off-target sites (OT1 and OT3) in HEK293T cells. The dashed bars represent the TIDE background. (C) Genome-wide specificity of VEsiCas targeting the VEGFA site3 locus. Shown is GUIDE-seq analysis for the sgRNA targeting VEGFA site3 locus in HEK293T cells transfected with SpCas9 or treated with VEsiCas. The black square indicates on-target sites. DNA from three biological replicates was mixed before library preparation. Right: the total number of off-targets identified for the sgVEGFA site3 with SpCas9 transfection or VEsiCas treatments.
Figure 4 Analysis of On/Off-Target Activity Generated by VEsiCas on the VEGFA Locus (A) Traceless delivery of SpCas9 through VEsiCas. Shown is a time course analysis of SpCas9 intracellular levels by western blot analysis in HEK293T cells transduced with VEsiCas or transfected with a plasmid expressing SpCas9. The reported time points correspond to the time of analysis following treatments (transduction or transfection). The western blot is representative of n = 2 independent experiments. (B) Targeted comparison of genome editing specificity using VEsiCas or a plasmid expressing SpCas9 in combination with sgVEGFA site3. Shown are percentages of indel formation measured by TIDE in the VEGFA site3 locus and in two sgVEGFA site3 previously validated off-target sites (OT1 and OT3) in HEK293T cells. The dashed bars represent the TIDE background. (C) Genome-wide specificity of VEsiCas targeting the VEGFA site3 locus. Shown is GUIDE-seq analysis for the sgRNA targeting VEGFA site3 locus in HEK293T cells transfected with SpCas9 or treated with VEsiCas. The black square indicates on-target sites. DNA from three biological replicates was mixed before library preparation. Right: the total number of off-targets identified for the sgVEGFA site3 with SpCas9 transfection or VEsiCas treatments.CC BY-NC-ND · PubMed Central

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Metadata

Authors
Montagna, C., Petris, G., Casini, A., Maule, G., Franceschini, G. M., Zanella, I., Conti, L., Arnoldi, F., Burrone, O. R., Zentilin, L., Zacchigna, S., Giacca, M., & Cereseto, A.
Affiliations
  • University of Trento (IT)
  • University of Trieste (IT)
  • AREA Science Park (IT)
Corresponding author
  • Petris, Gianluca
  • Cereseto, Anna
ORCID
Venue
Molecular Therapy Nucleic Acids
Publisher
Elsevier BV
Published
2018-09
Volume
12
Pages
453-462
ISSN
2162-2531 (ISSN-L)
DOI
10.1016/j.omtn.2018.05.010
PMC
PMC6041463
OpenAlex
W2874925241
Cited by
117
References
51
Citations by year
2026: 6 · 2025: 12 · 2024: 13 · 2023: 18 · 2022: 24 · 2021: 20 · 2020: 13 · 2019: 10 · 2018: 1
FWCI
3.91
Type
journal article
Language
en
Open access
Licence
Locations
  • Molecular Therapy — Nucleic Acids (publishedVersion, cc-by-nc-nd)
  • Europe PMC (PubMed Central) (publishedVersion, cc-by-nc-nd)
  • PubMed Central (publishedVersion, cc-by-nc-nd)
  • PubMed (publishedVersion)
  • ArTS Archivio della ricerca di Trieste (University of Trieste https://www.units.it/) (submittedVersion, cc-by-nc-nd)
  • Institutional Research Information System (Università degli Studi di Trento) (submittedVersion, cc-by-nc-nd)
  • Lirias (submittedVersion, cc-by-nc-nd)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
Funders
  • Università degli Studi di Trento
  • Fondazione Cassa Di Risparmio Di Trento E Rovereto
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › CRISPR and Genetic Engineering
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA Interference and Gene Delivery
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Virus-based gene therapy research
Keywords
CRISPR/Cas9 delivery, vesicular stomatitis virus glycoprotein, ribonucleoprotein complexes, single guide RNA, T7 RNA polymerase, off-target effects, genome editing, induced pluripotent stem cells, cardiomyocytes

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