Nanoparticle-Mediated Delivery of CRISPR/Cas9 Rnps Into Cells

The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPRassociated protein 9 (Cas9) system has revolutionized genome editing through its precision, simplicity, and programmability. The clinical translation of CRISPR/Cas9 Ribonucleoproteins (RNPs) remains constrained by major delivery challenges, including large molecular size, instability in biological fluids, and limited cellular penetration. Nanoparticle-mediated, non-viral delivery systems have emerged as a compelling strategy to overcome these barriers by providing targeted, transient, and biocompatible transport of Cas9 RNPs into cells. This review systematically evaluates recent advances in lipid-based, polymeric, peptide, and exosome-derived nanocarriers, focusing on their design principles, mechanisms of uptake, and endosomal escape strategies. Particular attention is given to receptor-mediated targeting, intracellular trafficking, and the emerging translation of lipid nanoparticle systems such as NTLA-2001, which exemplify clinical progress in genome editing. Despite these advancements, several challenges persist. Nanoparticle systems exhibit trade-offs between efficiency, biocompatibility, and scalability. For example, lipid nanoparticles may trigger anti- PEG immune reactions, exosome production faces manufacturing constraints, and polymeric systems risk cytotoxicity or limited degradability. Pre-existing immunity to Cas9 proteins and unpredictable immune responses to nanoparticle components remain significant translational hurdles. By integrating current preclinical and early clinical evidence, this review highlights both the promise and the limitations of nanoparticle-mediated CRISPR/Cas9 RNP delivery. Understanding these biological, immunological, and engineering challenges will be crucial for developing safe, efficient, and clinically scalable genome editing platforms.