We herein report an optogenetically activatable CRISPR-Cas9 nanosystem for programmable genome editing in the second near-infrared (NIR-II) optical window. The nanosystem, termed nanoCRISPR, is composed of a cationic polymer-coated Au nanorod (APC) and Cas9 plasmid driven by a heat-inducible promoter. The APC not only serves as a carrier for intracellular plasmid delivery but also can harvest external NIR-II photonic energy and convert it into local heat to induce the gene expression of the Cas9 endonuclease. Due to high transfection activity, the APC shows strong ability to induce a significant level of disruption in different genomic loci upon optogenetic activation. Moreover, the precise control of genome-editing activity can be simply programmed by finely tuning exposure time and irradiation time in vitro and in vivo and also enables editing at multiple time points, thus proving the sensitivity and inducibility of such an editing modality. The NIR-II optical feature of nanoCRISPR enables therapeutic genome editing at deep tissue, by which treatment of deep tumor and rescue of fulminant hepatic failure are demonstrated as proof-of-concept therapeutic examples. Importantly, this modality of optogenetic genome editing can significantly minimize the off-target effect of CRISPR-Cas9 in most potential off-target sites. The optogenetically activatable CRISPR-Cas9 nanosystem we have developed offers a useful tool to expand the current applications of CRISPR-Cas9, and also defines a programmable genome-editing strategy toward high precision and spatial specificity.
Keywords: CRISPR-Cas9; gene delivery; off target; photoregulation; spatiotemporal specificity.