This article demonstrates a fully synthetic strategy enabling CRISPR-mediated activation of tumour suppressor genes in vivo to reduce tumour burden.

Gold nanorods are one of the most widely explored inorganic materials in nanomedicine for diagnostics, therapeutics and sensing 1 . It has been shown that gold nanorods are not cytotoxic and localize within cytoplasmic vesicles following endocytosis, with no nuclear localization 2,3 , but other studies have reported alterations in gene expression profiles in cells following exposure to gold nanorods, via unknown mechanisms 4 . In this work we describe a pathway that can contribute to this phenomenon. By mapping the intracellular chemical speciation process of gold nanorods, we show that the commonly used Au-thiol conjugation, which is important for maintaining the noble (inert) properties of gold nanostructures, is altered following endocytosis, resulting in the formation of Au(i)-thiolates that localize in the nucleus 5 . Furthermore, we show that nuclear localization of the gold species perturbs the dynamic microenvironment within the nucleus and triggers alteration of gene expression in human cells. We demonstrate this using quantitative visualization of ubiquitous DNA G-quadruplex structures, which are sensitive to ionic imbalances, as an indicator of the formation of structural alterations in genomic DNA.Traditionally, gold nanorods (GNRs) are synthesized with a non-covalently bound bilayer of cetyltrimethylammonium bromide (CTAB) that dissociates from the GNR surface under physiological conditions, resulting in significant cytotoxicity. This can be overcome by exchanging CTAB with a thiolated analogue-(16-mercaptohexadecyl)trimethylammonium bromide (MTAB)-to enable covalent interaction between the gold surface and pendant thiol groups resulting in highly efficient non-toxic cellular internalization 6 . In this study, we synthesized two distinct MTAB-modified GNRs of different lengths (54.1 ± 5.2 nm and 91.8 ± 11.3 nm) and identical diameters (19.0 ± 2.0 nm and 18.9 ± 2.4 nm) with corresponding aspect ratios (ARs) of 2.8 and 4.9, respectively ( Fig. 1b-d). We also synthesized MTAB-coated gold nanoparticles (GNPs, Fig. 1a) of similar diameter to the GNRs as a control to evaluate shapedependent contributions. The complete exchange of CTAB for its thiolated analogue MTAB was validated by the detection of sulfur using electron energy loss spectra (EELS) ( Supplementary Fig. 1). The zeta potentials of the GNPs/GNRs were 32.8 ± 0.6 (GNP), 36.8 ± 0.6 (AR 2.8) and 25.8 ± 1.2 (AR 4.9) ( Supplementary Fig. 2). The UV-vis absorbance of the GNRs demonstrated a characteristic redshift in the longitudinal surface plasmon resonance (LSPR) from 679 to 953 nm for the short (NRS) and long (NRL) nanorods, respectively (Fig. 1e). The presence of the sharp LSPR for the GNRs (full-width at half-maximum (FWHM) = 106.9 nm (AR 2.8) and 195.0 nm (AR 4.9)) is indicative of their narrow size distribution. Both GNR samples had negligible amounts of shape impurities (for example, spheres) (Fig. 1b,c).We next evaluated the toxicity profile and cellular internalization of the GNRs in HEK-293T and MCF-7 cells. The toxicity profiles included...

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