Fig. 3: Strategies for reducing background signals to achieve higher contrast.

As an alternative way to enhance the S/N or S/B ratio, the CRISPR–dCas9 system uses various approaches to reduce background fluorescence. a A multitargeting gRNA was designed to deplete background fluorescence from freely diffusing dCas9–EGFP molecules. This gRNA has over 300,000 potential binding sites across the genome, ensuring that most dCas9–EGFP molecules are stably bound to DNA rather than diffusing, enabling the detection of single dCas9–EGFP molecules. b A split GFP strategy was implemented using an engineered dCas9–SunTag and sgRNA–MS2 system. GFP fragments (GFP1–9, scFv–GFP10 and MCP–GFP11) assemble into a functional fluorescent GFP only when the RNP complex binds to the target DNA. Diffusing fragments remain nonfluorescent, effectively reducing background fluorescence. c A MB approach was introduced, utilizing an sgRNA engineered to incorporate a MTS. The MB, colabeled with a fluorophore and a quencher, remains quenched when unbound due to the close proximity of the fluorophore and quencher, effectively minimizing background signals. d A LEXY was combined with the dCas9–SunTag imaging system and scFv–sfGFP fusion proteins. Upon CRISPR complex binding to the target region, LEXY is activated during imaging to export unbound scFv–sfGFP molecules out of the nucleus, reducing background fluorescence. e A protein destabilizing motif (tDeg) was fused to the FP, which is stabilized only when bound to a specific RNA aptamer, Pepper. Unbound FP–tDeg molecules are rapidly degraded via the tDeg-mediated signaling pathway, leading to a drastic reduction in background fluorescence.