Photoactivatable Engineering of CRISPR/Cas9-Inducible DNAzyme Probe for In Situ Imaging of Nuclear Zinc Ions

Abstract

DNAzyme-based fluorescent probes for imaging metal ions in living cells have received much attention recently. However, employing in situ metal ions imaging within subcellular organelles, such as nucleus, remains a significant challenge. We developed a three-stranded DNAzyme probe (TSDP) that contained a 20-base-pair (20-bp) recognition site of a CRISPR/Cas9, which blocks the DNAzyme activity. When Cas9, with its specialized nuclear localization function, forms an active complex with sgRNA within the cell nucleus, it cleaves the TSDP at the recognition site, resulting in the in situ formation of catalytic DNAzyme structure. With this design, the CRISPR/Cas9-inducible imaging of nuclear Zn²⁺ is demonstrated in living cells. Moreover, the superiority of CRISPR-DNAzyme for spatiotemporal control imaging was demonstrated by integrating it with photoactivation strategy and Boolean logic gate for dynamic monitoring nuclear Zn²⁺ in both HeLa cells and mice. Collectively, this conceptual design expands the DNAzyme toolbox for visualizing nuclear metal ions and thus provides new analytical methods for nuclear metal-associated biology.

Keywords: CRISPR; DNAzyme; Fluorescence probes; Nuclear Zinc Ions; Photoactivation.