Application of ALFA-tag and tyramide-based fluorescence signal amplification to expand the CRISPR-based DNA imaging toolkit v1

Understanding the spatial organization of genomes within chromatin is crucial for deciphering gene regulation. In this protocol we describe application of an ALFA-tagged dCas9 protein in combination with aA. thaliana centromere-specific gRNA and a fluorescence-labeled NbALFA nanobody to label centromere-specific sequences in fixed Arabidopsis thaliana nuclei. 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. Furthermore, we describe the combination of the highly sensitive Tyramide Signal Amplification (TSA) method with CRISPR-FISH.