VSV-G-Enveloped Vesicles for Traceless Delivery of CRISPR-Cas9
Open accessAbstract
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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The HDR pathway is not highly active in human cells, limiting clinical efficacy of HDR-based CFTR repair.
“Nevertheless, the HDR pathway is not highly active in human cells, thus strongly limiting the clinical efficacy of this application23,24.”
From Allele specific repair of splicing mutations in cystic fibrosis through AsCas12a genome editing (2019) · cites this paper as [23]
The Results (Design and Development of VEsiCas; page unavailable) report HDR-mediated repair of an EGFP reporter in cultured cells, but do not say that HDR has low activity in human cells. The cited paper’s passages do not address CFTR repair or link HDR activity to its clinical efficacy (searched: HDR, CFTR, clinical efficacy). (LLM, conf=0.73)
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- 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)
- 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
- gold
- doi.org
- 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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