Iron‐Confined CRISPR/Cas9‐Ribonucleoprotein Delivery System for Redox‐Responsive Gene Editing

Abstract Clustered regularly interspaced short palindromic repeat (CRISPR)‐associated protein 9 (Cas9) is a promising gene editing tool to treat diseases at the genetic level. Nonetheless, the challenge of the safe and efficient delivery of CRISPR/Cas9 to host cells constrains its clinical applicability. In the current study, a facile, redox‐responsive CRISPR/Cas9‐Ribonucleoprotein (RNP) delivery system by combining iron‐coordinated aggregation with liposomes (Fe‐RNP@L) is reported. The Fe‐RNP is formed by the coordination of Fe³⁺ with amino and carboxyl groups of Cas9, which modifies the lipophilicity and surface charge of RNP and alters cellular uptake from primary endocytosis to endocytosis and cholesterol‐dependent membrane fusion. RNP can be rapidly and reversibly released from Fe‐RNP in response to glutathione without loss of structural integrity and enzymatic activity. In addition, iron coordination also improves the stability of RNP and substantially mitigates cytotoxicity. This construct enabled highly efficient cytoplasmic/nuclear delivery (≈90%) and gene‐editing efficiency (≈70%) even at low concentrations. The high payload content, high editing efficiency, good stability, low immunogenicity, and ease of production and storage, highlight its potential for diverse genome editing and clinical applications.