Nitroreductase-Responsive Oligomeric crRNAs for Enzyme-Triggered Regulation of CRISPR Activity

Hypoxic tumors overexpress nitroreductase (NTR), providing an endogenous trigger for selective biomolecular activation. Here, we describe the synthesis of NTR-responsive clustered regularly interspaced short palindromic repeats (CRISPR) guide RNAs via the site-specific incorporation of a p -nitrobenzyl ( p -NB) phosphoramidite at the 5′ terminus of crRNAs. Click-mediated oligomerization into trimeric and tetrameric constructs effectively suppressed Cas nuclease activity. Enzymatic reduction by NTR induced linker cleavage, releasing active crRNAs and restoring DNA cleavage in vitro, establishing a strategy for enzyme-regulated CRISPR control.