The potential of ALFA-tag and tyramide-based fluorescence signal amplification to expand the CRISPR-based DNA imaging toolkit.

Understanding the spatial organization of genomes within chromatin is crucial for deciphering gene regulation. A recently developed CRISPR-dCas9-based genome labeling tool, known as CRISPR-FISH, allows efficient labelling of repetitive sequences. Unlike standard fluorescence in situ hybridization (FISH), CRISPR-FISH eliminates the need for global DNA denaturation, allowing for superior preservation of chromatin structure. Here, we report on the further development of the CRISPR-FISH method, which has been enhanced for increased efficiency through the engineering of a recombinant dCas9 protein containing an ALFA-tag. Using an ALFA-tagged dCas9 protein assembled with an A. thaliana centromere-specific gRNA, we demonstrate target-specific labelling with a fluorescence-labeled NbALFA nanobody. The dCas9 protein possessing multiple copies of the ALFA-tag, in combination with a minibody and fluorescence-labelled anti-rabbit secondary antibody, resulted in enhanced target-specific signals. The dCas9-ALFA-tag system was also instrumental in live cell imaging of telomeres in N. benthamiana. This method will further expand the CRISPR imaging toolkit, facilitating a better understanding of genome organization. Furthermore, we report the successful integration of the highly sensitive Tyramide Signal Amplification (TSA) method with CRISPR-FISH, demonstrating effective labeling of A. thaliana centromeres.