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.

SUBMITTER: Potlapalli BP 

PROVIDER: S-EPMC11522987 | biostudies-literature | 2024 Oct

REPOSITORIES: biostudies-literature