The CRISPR/Cas system has a broad range of possible medical applications. However, its translation towards clinical use has been hampered due to e.g., multiple safety issues like unpredictable off-targets related side effects. Improved safety can be achieved by the delivery of CRISPR-Cas9 to target cells in form of ribonucleoprotein complexes (RNP). To this goal we took advantage of extracellular vesicles (EV) abundance and versatility to carry and deliver Cas9-RNPs. We used different types of EV to mediate genome editing in eukaryotic cells with a particular focus on virus-like-particles (VLP). However, biochemical, structural, and functional knowledge, which is essential for secure handling of VLPs, is still needed.

Our goal here is to develop a new pipeline that would allow us to characterize and compare different types of Cas-RNPs preloaded vesicles, leading to efficient and safe genome editing. For this reason, we generate gammaretrovirus and lentivirus-derived particles targeting GFP expressed by reporter cell lines. We optimize the production process by determining their size and concentration by Nanosight (NTA) and quantifying Cas9 protein by enzyme-linked immunosorbent assay (ELISA). We demonstrate we could predict the level of genome editing and correlate it to the EV concentration and their Cas9 load. Additionally, we used high resolution imaging flow cytometry (Imagestream Mark II) to characterize homogeneity of the population of both VLP-types, by EV-markers staining (tetraspanins family) to identify different subclasses of EVs in our VLP supernatants.