Engineering Nucleotidoproteins for Base‐Pairing‐Assisted Cytosolic Delivery and Genome Editing

Abstract Protein therapeutics targeting intracellular machineries hold profound potential for disease treatment, and hence robust cytosolic protein delivery technologies are imperatively demanded. Inspired by the super‐negatively charged, nucleotide‐enriched structure of nucleic acids, adenylated pro‐proteins (A‐proteins) with dramatically enhanced negative surface charges have been engineered for the first time via facile green synthesis. Then, thymidine‐modified polyethyleneimine is developed, which exhibits strong electrostatic attraction, complementary base pairing, and hydrophobic interaction with A‐proteins to form salt‐resistant nanocomplexes with robust cytosolic delivery efficiencies. The acidic endolysosomal environment enables traceless restoration of the A‐proteins and consequently promotes the intracellular release of the native proteins. This strategy shows high efficiency and universality for a variety of proteins with different molecular weights and isoelectric points in mammalian cells. Moreover, it enables highly efficient delivery of CRISPR‐Cas9 ribonucleoproteins targeting fusion oncogene EWSR1‐FLI1, leading to pronounced anti‐tumor efficacy against Ewing sarcoma. This study provides a potent and versatile platform for cytosolic protein delivery and gene editing, and may benefit the development of protein pharmaceuticals.