Direct cellular delivery of CRISPR/Cas9 complexes is of great significance for genome editing and other recently developed applications, such as gene expression regulation and RNA/DNA imaging. Here, we first constructed a graphene oxide (GO)-polyethylene glycol (PEG)-polyethylenimine (PEI) nanocarrier for the delivery of high-molecular-weight Cas9/single-guide RNA (sgRNA) complexes for endocytosis, endosomal escape, nuclear entry, and gene editing. The results demonstrate that the nanocarrier can be used successfully for efficient gene editing in human AGS cells with an efficiency of ∼39%. The results also show that this nanocarrier can protect sgRNA from enzymatic degradation, thus exhibiting extremely high stability, which is critical for future in vivo applications. Thus, this GO-mediated Cas9/sgRNA delivery system has potential as a new approach for biomedical research and targeted gene engineering applications.
Near-infrared (NIR)-light-triggered nanomedicine, including photodynamic therapy (PDT) and photothermal therapy (PTT), is growing an attractive approach for cancer therapy due to its high spatiotemporal controllability and minimal invasion, but the tumor eradication is limited by the intrinsic anti-stress response of tumor cells. Herein, we fabricate a tumor-microenvironment responsive CRISPR nanoplatform based on oxygen-deficient titania (TiO 2- x ) for mild NIR-phototherapy. In tumor microenvironment, the overexpressed hyaluronidase (HAase) and glutathione (GSH) can readily destroy hyaluronic acid (HA) and disulfide bond and releases the Cas9/sgRNA from TiO 2- x to target the stress alleviating regulators, i.e. , nuclear factor E2-related factor 2 (NRF2) and heat shock protein 90 α (HSP90 α ), thereby reducing the stress tolerance of tumor cells. Under subsequent NIR light illumination, the TiO 2- x demonstrates a higher anticancer effect both in vitro and in vivo . This strategy not only provides a promising modality to kills cancer cells in a minimal side-effects manner by interrupting anti-stress pathways but also proposes a general approach to achieve controllable gene editing in tumor region without unwanted genetic mutation in normal environments.
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