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

Abstract

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 labeling 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 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 Arabidopsis centromere-specific guide RNA, we demonstrate target-specific labeling with a fluorescence-labeled NbALFA nanobody. The dCas9 protein possessing multiple copies of the ALFA-tag, in combination with a minibody and fluorescence-labeled 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 Nicotiana 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 method with CRISPR–FISH, demonstrating effective labeling of Arabidopsis centromeres.