Fluorogenic CRISPR for genomic DNA imaging.
Nat Commun
; 15(1): 934, 2024 Jan 31.
Article
en En
| MEDLINE
| ID: mdl-38296979
ABSTRACT
Genomic DNA exhibits high heterogeneity in terms of its dynamic within the nucleus, its structure and functional roles. CRISPR-based imaging approaches can image genomic loci in living cells. However, conventional CRISPR-based tools involve expressing constitutively fluorescent proteins, resulting in high background and nonspecific nucleolar signal. Here, we construct fluorogenic CRISPR (fCRISPR) to overcome these issues. fCRISPR is designed with dCas9, an engineered sgRNA, and a fluorogenic protein. Fluorogenic proteins are degraded unless they are bound to specific RNA hairpins. These hairpins are inserted into sgRNA, resulting in dCas9 sgRNA fluorogenic protein ternary complexes that enable fluorogenic DNA imaging. With fCRISPR, we image various genomic DNA in different human cells with high signal-to-noise ratio and sensitivity. Furthermore, fCRISPR tracks chromosomes dynamics and length. fCRISPR also allows DNA double-strand breaks (DSBs) and repair to be tracked in real time. Taken together, fCRISPR offers a high-contrast and sensitive platform for imaging genomic loci.
Texto completo:
1
Base de datos:
MEDLINE
Asunto principal:
Sistemas CRISPR-Cas
/
ARN Guía de Sistemas CRISPR-Cas
Idioma:
En
Revista:
Nat Commun
Asunto de la revista:
BIOLOGIA
/
CIENCIA
Año:
2024
Tipo del documento:
Article