Fig. 6: CRISPR–dCas9 technologies for fixed-cell imaging.
a The binding of the RNP complex to target sites does not require a denaturation step for dsDNA. By applying a preassembled fluorescent RNP complex to fixed specimens, both repetitive and nonrepetitive genomic regions can be readily visualized using single or multiple sgRNAs, respectively, without disrupting chromatin organization. b The introduction of a Cas9 nickase (Cas9dHNH) and a superhelicase (Rep-X) enables local denaturation of dsDNA, facilitating the binding of conventional DNA-FISH probes. This approach allows any genomic region to be visualized with higher labeling density, free from the constraints of PAM sequences. c Signal amplification via an exchange reaction has been used to image nonrepetitive sequences using a single sgRNA. An engineered sgRNA containing a PER-binding site can recruit a PER concatemer, which can subsequently be labeled with PER probes in an iterative manner, enhancing signal intensity drastically. d, e SNP sites in fixed cells were efficiently detected using RCA. Two RNP complexes were designed to target genomically proximal regions, with one region potentially harboring an SNP mutation at the third position from the PAM. Each sgRNA was engineered to include a distinct hairpin structure, allowing recognition by corresponding probes. d A proximity probe was introduced, designed to bind the target region only when the two RNP complexes are correctly positioned. RCA elongates the probe tethered to the RNP complex at the potential SNP site, enabling visualization through fluorescent probe binding. SNP mutations disrupt the binding of one RNP complex, preventing RCA and eliminating the fluorescent signal. e Alternatively, distinct hairpin probes can initiate separate RCA reactions, removing the proximity restriction for the two RNP complexes. In this setup, SNP mutations result in the loss of one RCA reaction, enabling analysis by measuring the loss of colocalization between the signals.
