Chemical Evolution of Amphiphilic Xenopeptides for Potentiated Cas9 Ribonucleoprotein Delivery

[Image: see text] The introduction of the CRISPR/Cas9 system in the form of Cas9/sgRNA ribonucleoproteins (RNP) is an efficient, straightforward strategy for genome editing, and potent RNP carriers are in high demand. Here, we report a series of artificial peptides based on novel ionizable amino acids that are able to deliver Cas9 RNP into cells very efficiently. Systematic variation of hydrophobic properties revealed a relationship between the xenopeptide logD(7.4) and genome editing potency. By correlating the physicochemical properties with biological activity, individual optima were found for different xenopeptide sequence architectures. The optimized amphiphilic carriers enable ∼88% eGFP knockout at an RNP dose of only 1 nM and up to 40% homology-directed repair (HDR) in eGFP/BFP switchable reporter cells by co-delivery with an ssDNA template. Mechanistic studies demonstrated that hydrophobically balanced xenopeptides are more resistant to ionic stress as well as concentration-dependent dissociation and promote endocytosis by both clathrin- and macropinocytosis-mediated pathways. The systematic study develops a versatile and adjustable carrier platform and highlights impactful structure–activity relationships, providing a new chemical guide for the design and optimization of nonviral Cas9 RNP nanocarriers.