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The CRISPR/Cas system is a powerful tool for both scientific research and potential therapeutic strategies for inherited diseases. This PhD thesis investigated the feasibility of cell-penetrating peptide-mediated direct delivery of CRISPR/Cas components to achieve various CRISPR/Cas-based gene editing applications. In order to reduce off-target effects that result from long-term high expression and activity of CRISPR/Cas9, rather than employing mRNA or plasmid platforms of Cas9 as delivery format, this work focused on the direct delivery of the Cas9 ribonucleoprotein (RNP) complex. After screening a variety of cell-penetrating peptide sequences, we encovered a highly efficient delivery of Cas9 using the amphipathic LAH5 peptide. This peptide sequence showed a robust capacity to form complexes with Cas9 ribonucleoprotein, even with an additional single-stranded DNA homology-directed repair (HDR) template which can be used to generate specific genetics repairs or mutations. The nanocomplexes formed with the Cas9 ribonucleoprotein and the LAH5 peptides were effectively taken up by cells, resulting in effective gene editing and correction in a variety of human cell types. Whereas cell-penetrating peptides have been shown to facilitate efficient transfected of various cargos in an in vitro setting, these systems have various limitations that may hamper therapeutic applications, such as decreased stability in the presence of serum. To address these limitations, we studied the effects of N-terminal acylation with various fatty acids on the capacity of LAH5 peptides to form nanocomplexes with Cas9 ribonucleoprotein. Furthermore, we studied the overall stability of such nanocomplexes, as well as their protective capabilities against protein-degrading enzymes, their capability of disrupting cell membranes, and their efficiency in Cas9 delivery. Fatty acid modifications, especially Oleic acid provided increased durability against proteases and increased stability in serum-rich

Peptides able to translocate cell membranes while carrying macromolecular cargo, as cell-penetrating peptides (CPPs), can contribute to the field of drug delivery by enabling the transport of otherwise membrane impermeable molecules. Formation of non-covalent complexes between amphipathic peptides and oligonucleotides is driven by electrostatic and hydrophobic interactions. Here we investigate and quantify the coexistence of distinct molecular species in multiple equilibria, namely peptide monomer, peptide self-aggregates and peptide/oligonucleotide complexes. As a model for the complexes, we used a stearylated peptide from the PepFect family, PF14 and siRNA. PF14 has a cationic part and a lipid part, resembling some characteristics of cationic lipids. Fluorescence correlation spectroscopy (FCS) and fluorescence cross-correlation spectroscopy (FCCS) were used to detect distinct molecular entities in solution and at the plasma membrane of live cells. For that, we labeled the peptide with carboxyrhodamine 6G and the siRNA with Cyanine 5. We were able to detect fluorescent entities with diffusional properties characteristic of the peptide monomer as well as of peptide aggregates and peptide/oligonucleotide complexes. Strategies to avoid peptide adsorption to solid surfaces and self-aggregation were developed and allowed successful FCS measurements in solution and at the plasma membrane. The ratio between the detected molecular species was found to vary with pH, peptide concentration and the proximity to the plasma membrane. The present results suggest that the diverse cellular uptake mechanisms, often reported for amphipathic CPPs, might result from the synergistic effect of peptide monomers, self-aggregates and cargo complexes, distributed unevenly at the plasma membrane.