High-Efficiency Tracible Nanodiamond-Mediated Gene Editing System Development and Application

Nanodiamonds (NDs) are biocompatible crystalline carbon nanomaterials for biomolecules or small molecules delivery into the target cells especially for NDs smaller than 5 nm. By conjugating NDs with red fluorescence protein, mCherry, the distribution of these nano-fluorescence nanoparticles (ND-mCherry) can be traced in the cells or tissues. Moreover, by conjugating linear DNA fragments with all the components of the CRISPR/Cas9 genome editing system, the NDs particles became a gene-editing device. X-linked retinoschisis (XLRS) is an inherited retinal disorder disease that leads to reduced visual acuity in affected persons. Genetic analysis indicated that XLRS is caused by mutations c.625T in the RS1 gene. Therefore, RS1 serves as a target for gene editing systems. The low transfection efficiency is one of the major challenges of conventional CRISPR/Cas9 genomic editing systems. In this study, we developed an ND-mCherry-CRISPR / Cas9 gene editing system (ND-mCherry-CRISPR / Cas9). The size of ND is approximately 3 nm, which contained both a target gene sequence and an extra green fluorescence protein gene as a gene expression reporter. This tracible ND-mediated gene editing system is not only delivered genes into hiPSCs and mouse retina but also mediates gene editing events. Therefore, the structure of the mouse photoreceptor layer was loose, and cone/rod cells were also found to be abnormal by the ND-mediated gene editing system introduced. The effective gene editing efficiency is about 10 times higher than conventional CRISPR / Cas9. Namely, ND-mediated gene editing systems can be used as a way to quickly establish animal models of hereditary diseases. Furthermore, by conjugating with the gene correction sequences and our system will be a potential medical device for disease treatment.