Discussion

CRISPR/Cas9 technology has revolutionized the field of cell biology, introducing efficient genome editing into the toolbox 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 delivery of Cas9 protein is as effective as plasmid-based delivery, but with the added benefit of fewer off-target effects due to the short lifespan of the Cas9 protein in the cell (1). Here we report delivery of Cas9 protein using cell-derived nanovesicles called gesicles. Gesicles are produced by a mammalian packaging cell via co-overexpression of three components: a nanovesicle-inducing glycoprotein, Cas9, and the sgRNA specific to the target gene. Gesicles display a protein on their surface that mediates binding and fusion with the cellular membrane of target cells. Based on this principle, we have developed a method for actively packaging sgRNA-loaded Cas9 into gesicles via ligand-dependent dimerization(iDimerize™ technology). This dimerization approach allowed us to efficiently package active Cas9 containing a nuclear localization signal (NLS) into these nanovesicles. Gesicle-based protein delivery does not rely on recombinant protein from a bacterial source or the use of a transfection reagent. We show that gesicles carrying a Cas9-sgRNA protein complex (Cas9 Gesicles) can mediate target-gene knockout in a variety of cell types. The observed knockout efficiency is often considerably higher than what is observed with plasmid-based delivery of sgRNA and Cas9. This nanoparticle-based method allows for tight control of the dose and duration of the Cas9-sgRNA complex in the cell; this level of control decreases off-target effects. Close analysis of the particles shows that they are stable over several freeze/thaw cycles and are consistent in size (~150–170 nm). Overall, these particles can be considered a novel and effective tool for Cas9 and sgRNA delivery to any target cells.