Biomimetic Mineralized CRISPR/Cas RNA Nanoparticles forEfficient Tumor-Specific Multiplex Gene Editing

CRISPR/Cas9 systems have great potential to achieve sophisticated gene therapy and cell engineering by editing multiple genomic loci. However, to achieve efficient multiplex gene editing, the delivery system needs adequate capacity to transfect all CRISPR/Cas9 RNA species at the required stoichiometry into the cytosol of each individual cell. Herein, inspired by biomineralization in nature, we develop an all-in-one biomimetic mineralized CRISPR/Cas9 RNA delivery system. This system allows for precise control over the coencapsulation ratio between Cas9 mRNA and multiple sgRNAs, while also exhibiting a high RNA loading capacity. In addition, it enhances the storage stability of RNA at 4 °C for up to one month, and the surface of the nanoparticles can be easily functionalized for precise targeting of RNA nanoparticles in vivo at nonliver sites. Based on the above characteristics, as a proof-of-concept, our system was able to achieve significant gene-editing at each target gene (Survivin: 31.9%, PLK1: 24.41%, HPV: 23.2%) and promote apoptosis of HeLa cells in the mouse model, inhibiting tumor growth without obvious off-target effects in liver tissue. This system addresses various challenges associated with multicomponent RNA delivery in vivo, providing an innovative strategy for the RNA-based CRISPR/Cas9 gene editing.