FAST‐CRISPR: Fusogenic Association and Secured Transfection of CRISPR/Cas9 Ribonucleoproteins Using Lipid‐Silica Hybrid Nanoparticles for Therapeutic Genome Editing

ABSTRACT Clinical translation of CRISPR/Cas9 therapeutics is challenged by inefficient cytosolic delivery and toxicity issues associated with viral vectors and nanoparticle‐based carriers. To overcome these concerns, herein we report a lipid‐silica hybrid nanoparticle platform for fusogenic association and secured transfection of CRISPR/Cas9 (FAST‐CRISPR), designed for rapid cytosolic delivery of CRISPR/Cas9 ribonucleoproteins, followed by efficient gene editing. Through direct fusion with the plasma membrane and bypassing conventional endocytic barriers, FAST‐CRISPR nanoparticles displayed superior intracellular delivery efficacy. Optimizing lipid compositions, we discovered that a 1:1 weight mixture of cationic DOTAP and ionizable DODMA lipids, combined with tailored large‐pore silica nanoparticles, enables enhanced loading capacity, rapid cytosolic dispersion, and significant nuclear transport of Cas9/gRNA complexes. FAST‐CRISPR nanoparticles efficiently delivered multiplex genome‐targeting ribonucleoproteins to induce targeted double‐strand DNA breaks, triggering apoptosis in cancer cells and significantly suppressing tumor growth in a mouse xenograft model without systemic toxicity. Our findings demonstrate the therapeutic efficacy and translational potential of FAST‐CRISPR nanoparticles as a safe and versatile non‐viral delivery platform for precision genome editing.