Successful and efficient delivery of Cas9 protein and gRNA into cells is critical for genome editing and its therapeutic application. In this study, we developed an improved supercharged polypeptide (SCP) mediated delivery system based on dithiocyclopeptide linker to realize the effective genome editing in tumor cells. The fusion protein Cas9-linker-SCP (Cas9-LS) forms positively charged complexes with gRNA in vitro to provide possibilities for gRNA delivery into cells. Under the microenvironment of tumor cells, the dithiocyclopeptide linker, containing matrix metalloproteinase 2 (MMP-2) sensitive sequence and an intramolecular disulfide bond, can be completely disconnected to promote the release of Cas9 protein with the nuclear localization sequence (NLS) in the cytoplasm and transfer to the cell nucleus for highly efficient genome editing, resulting in an obvious increase of indel efficiency in comparison to fusion protein without dithiocyclopeptide linker (Cas9-SCP). Furthermore, Cas9-LS shows no significant cytotoxicity and minimal hemolytic activity. We envision that the microenvironment-responsive Cas9 protein delivery system can facilitate more efficient genome editing in tumor cells.

Despite the well-recognized clinical success of therapeutic proteins, especially antibodies, their inability to penetrate the cell membrane restricts them to secretory extracellular or membrane-associated targets. Developing a direct cytosolic protein delivery system would offer unique opportunities for intracellular target-related therapeutic proteins. Here, we generated a supercharged polypeptide (SCP) with high cellular uptake efficiency, endosomal escape ability, and good biosafety and developed an SCP with an unnatural amino acid containing the phenylboronic acid (PBA) group, called PBA-SCP. PBA-SCP is capable of potently delivering proteins with various isoelectric points and molecular sizes into the cytosol of living cells without affecting their bioactivities. Importantly, cytosolically delivered antibodies remain functional and are capable of targeting, labeling, and manipulating diverse intracellular antigens. This study demonstrates an efficient and versatile intracellular protein delivery platform, especially for antibodies, and provides new possibilities for expanding protein-based therapeutics to intracellular “undruggable” targets.

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