Lipopeptide‐Based Nanosome‐Mediated Delivery of Hyperaccurate CRISPR/Cas9 Ribonucleoprotein for Gene Editing

A transient cytosolic delivery system for accurate Cas9 ribonucleoprotein is a key factor for target specificity of the CRIPSR/Cas9 toolkit. Owing to the large size of the Cas9 protein and a long negative strand RNA, the development of the delivery system is still a major challenge. Here, a size‐controlled lipopeptide‐based nanosome system is reported, derived from the blood‐brain barrier‐permeable dNP2 peptide which is capable of delivering a hyperaccurate Cas9 ribonucleoprotein complex (HypaRNP) into human cells for gene editing. Each nanosome is capable of encapsulating and delivering ≈2 HypaRNP molecules into the cytoplasm, followed by nuclear localization at 4 h post‐treatment without significant cytotoxicity. The HypaRNP thus efficiently enacts endogenous eGFP silencing and editing in human embryonic kidney cells (up to 27.6%) and glioblastoma (up to 19.7% frequency of modification). The lipopeptide‐based nanosome system shows superior delivery efficiency, high controllability, and simplicity, thus providing biocompatibility and versatile platform approach for CRISPR‐mediated transient gene editing applications.