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

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.

CRISPR-Cas9 Dual-gRNA Attack Causes Mutation, Excision and Inversion of the HIV-1 Proviral DNA

“…A sterilizing cure would require that all HIV-infected cells are targeted with the gRNA and Cas9 components, but reducing the viral load below a certain level may suffice (functional cure). Several delivery methods are available, e.g., for transient editing with gRNA-Cas9 ribonucleoprotein particles or virus-like particles [48][49][50], and for durable editing with adeno-associated virus or lentiviral vectors [17,21,51], but these methods may not reach sufficient infected cells in vivo. Novel delivery methods may be needed that specifically target HIV-reservoir cells, possibly facilitated by a specific marker like CD32a [52,53].…”

Non-viral delivery of CRISPR/Cas9 complex using CRISPR-GPS nanocomplexes

“…In order to address these challenges, transient delivery of sgRNA along with the Cas9 mRNA or Cas9 protein has been achieved using non-viral methods such as electroporation, 15,16 hydrodynamic injection, 17 microinjection, 18 lipids, [19][20][21][22] peptides, [23][24][25][26][27][28] polyethylenimine in combination with other agents such as DNA nanoclew, graphene oxide, cholesterol, [29][30][31] gold nanoparticles and other nanostructures, [32][33][34][35][36][37][38] extracellular vesicles, [39][40][41][42] virus-like particles, 43 and biolistic delivery in plants. 44,45 Apart from these methods, many hybrid frameworks have also been used to deliver CRISPR/Cas9.…”

Extracellular nanovesicles for packaging of CRISPR-Cas9 protein and sgRNA to induce therapeutic exon skipping

“…Although EV-mediated CRISPR-Cas9 RNP delivery methods have been reported, such as Cas9P LV 28 and NanoBlades 29 systems that fuse SpCas9 with retroviral Gag, VEsiCas 30 system that passively incorporates SpCas9, or Gesicle 31 system that uses dimerization based incorporation of SpCas9, applicability for in vivo muscle tissue has not been elucidated. Furthermore, direct fusion of SpCas9 with Gag requires the supplementation of wildtype Gag-Pol to liberate Cas9 from Gag via protease-mediated cleavage, which competes for space within the EV and reduces the number of SpCas9 molecules packaged.…”