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Mol Ther Nucleic Acids. 2018 Sep 7;12:453-462. doi: 10.1016/j.omtn.2018.05.010. Epub 2018 Jul 11.

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

Author information

1
Laboratory of Molecular Virology, University of Trento, Centre for Integrative Biology, 38123 Trento, Italy.
2
Laboratory of Molecular Virology, University of Trento, Centre for Integrative Biology, 38123 Trento, Italy. Electronic address: gianluca.petris@unitn.it.
3
Laboratory of Stem Cell Biology, University of Trento, Centre for Integrative Biology, 38123 Trento, Italy.
4
International Centre for Genetic Engineering and Biotechnology (ICGEB), AREA Science Park, Padriciano 99, 34149 Trieste, Italy; Department of Medicine, Surgery and Health Sciences, University of Trieste, 34149 Trieste, Italy.
5
International Centre for Genetic Engineering and Biotechnology (ICGEB), AREA Science Park, Padriciano 99, 34149 Trieste, Italy.
6
Laboratory of Molecular Virology, University of Trento, Centre for Integrative Biology, 38123 Trento, Italy. Electronic address: anna.cereseto@unitn.it.

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.

KEYWORDS:

CRISPR-Cas9; GUIDE-seq; RNPs; SpCas9; VEsiCas; VLPs; genome editing; nuclease; ribonucleoprotein complex; viral-like particles

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