53We herein report the first optogenetically activatable CRISPR/Cas9 nanosystem for 54 programmable genome editing in the second near-infrared (NIR-II) optical window. The 55 nanosystem is composed of a cationic polymer-coated gold nanorod (APC) and Cas9 56 plasmid driven by a heat-inducible promoter. APC not only serves as a carrier for 57 intracellular plasmid delivery, but also can harvest external NIR-II photonic energy and 58 convert into local heat to induce the gene expression of Cas9 endonuclease. Due to high 59 transfection activity, APC shows strong ability to induce significant level of disruption in 60 different genome loci upon optogenetic activation. Moreover, the precise control of 61 genome editing activity can be simply programmed by finely tuning exposure time and 62 irradiation times in vitro and in vivo, and also enables editing at multiple time points, thus 63 proving the sensitivity and reversibility of such an editing modality. The NIR-II optical 64 feature of APC enables therapeutic genome editing at the deep tissue of the tumor-65 bearing mice, by which tumor growth could be effectively inhibited as a proof-of-concept 66 therapeutic example. Importantly, this modality of optogenetic genome editing can 67 significantly minimize the off-target effect of CRISPR/Cas9 in the most potential off-target 68 sites. The optogenetically activatable CRISPR/Cas9 nanosystem we have developed 69 offers a useful tool to expand the current applications of CRISPR/Cas9, and also defines 70 a programmable genome editing strategy towards unprecedented precision and 71 spatiotemporal specificity.
72
Introduction
73The RNA-guided clustered, regularly interspaced, short palindromic repeats (CRISPR)-74 associated nuclease protein 9 (Cas9) is originally an adaptive immune defense system, 75 by which many bacteria exploit to protect themselves from invading genetic elements 1 . It 76 has been recently harnessed as an efficient tool for genome editing in both single cells 77 and the whole organism for a wide range of biomedical applications in biology, genetics, 78 and medicine, etc 2,3 . In principle, the CRISPR/Cas9 is composed a single-guide RNA 79 (sgRNA) for the identification of DNA targets and a Cas9 endonuclease that can bind 80 and process the recognized DNA targets 4 . CRISPR/Cas9-based genome editing 81 technology offers a powerful and reliable strategy for the targeted modifications of the 82 genome, enabling the precise perturbation of virtually any genomic sequence in living 83 cells 2-6 . Due to its genome-wide specificity and multiplexing capability, Cas9 and its 84 variants have shown great potentials in the generation of loss-of-function animals 7,8 , the 85 correction of genetic disorders 9,10 , functional genome screening [11][12][13][14][15] , and the treatment of 86 infectious diseases 16,17 . Despite of these excitements, the lack of temporal and spatial 87 precision during editing process has severely constrained the current CRISPR/Cas9 88 systems from complicated and diverse genome-editing s...