Guanidyl-rich α-helical polypeptide enables efficient cytosolic pro-protein delivery and CRISPR-Cas9 genome editing.

Intracellular delivery of proteins has attracted significant interest in biological research and cancer treatment, yet it continues to face challenges due to the lack of effective delivery approaches. Herein, we developed an efficient strategy cationic α-helical polypeptide-mediated anionic proprotein delivery. The protein was reversibly modified with adenosine triphosphate dynamic covalent chemistry to prepare an anionic proprotein (A-protein) with abundant phosphate groups. A guanidyl-decorated α-helical polypeptide (LPP) was employed not only to encapsulate A-protein through electrostatic attraction and hydrogen bonding, forming stable nanocomplexes, but also to enhance cell membrane penetration due to its rigid α-helical conformation. Consequently, this strategy mediated the effective delivery of various proteins with different isoelectric points and molecular weights, including α-chymotrypsin, bovine serum albumin, ribonuclease A, cytochrome , saporin, horseradish peroxidase, β-galactosidase, and anti-phospho-Akt, into cancer cells. More importantly, it enabled efficient delivery of CRISPR-Cas9 ribonucleoproteins to elicit robust polo-like kinase 1 genome editing for inhibiting cancer cell growth. This rationally designed protein delivery system may benefit the development of intracellular protein-based cancer therapy.