Work

313. Gesicle Mediated Delivery of a Cas9-sgRNA Protein Complex

Year: 2016

Type: article

Abstract: CRISPR/Cas9-based gene editing has revolutionized the field of cell biology and is quickly being incorporated into the toolboxes of many laboratories. While CRISPR/Cas9 is a powerful technique for genome manipulation, two significant challenges remain: obtaining efficient delivery of Cas9 to all cell types and achieving fewer off-target effects. Recently, it has been demonstrated that genome editing via direct delivery of Cas9 is as effective as plasmid-based delivery, but with the added benefit of fewer off-target effects due to the short duration of the Cas9 protein in the cell (1). Here we report on the delivery of Cas9 using cell-derived nanovesicles termed gesicles. Gesicles are produced via co-overexpression of three components in a mammalian packaging cell: a nanovesicle-inducing glycoprotein, Cas9 endonuclease, and the sgRNA specific to the target gene. Additionally, we have developed a method for actively packaging sgRNA-loaded Cas9 into gesicles via ligand-dependent dimerization (iDimerize™ technology). The active packaging mechanism results in an approximate 4-fold increase in the loading of Cas9 into the gesicles. Moreover, the ligand-dependent dimerization approach also allowed us to efficiently package active Cas9 containing a nuclear localization signal (NLS) into these nanovesicles. We also have developed an optimized, lyophilized, one-step transfection formulation to promote high transfection efficiencies during the production of the Cas9-sgRNA gesicles. Gesicle-based protein delivery does not rely on recombinant protein production from a bacterial source or on the use of a transfection reagent for delivery. In this work, we were able to demonstrate that gesicles carrying a Cas9-sgRNA protein complex can mediate specific target-gene knockout in a wide variety of cell types, including human iPS cells. The observed knockout efficiency is often considerably higher than that observed with plasmid-based delivery of sgRNA and Cas9. This nanovesicle-based method allows for tight control of the dose and duration of the Cas9-sgRNA complex in the cell which has been shown to correlate with the amount of off-target cleavage (1). Through mismatch detection and Sanger sequencing of edited target sequences, we were able to demonstrate a lack of off-target cleavage when compared to plasmid-based delivery. Close analysis of the gesicles shows that they are stable over several freeze/thaw cycles and are consistent in size (~150-170 nm) as determined by nanoparticle tracking analysis. Overall, gesicles can be considered a novel and effective tool for simultaneous Cas9 and sgRNA delivery to any target cells. (1) Zuris, J. A. et al. (2014) Nat. Biotechnol. 33(1): 73-80.

Source: Molecular Therapy

DOI: 10.1016/s1525-0016(16)33122-7