CRISPR/Cas9-Based RGEN-ISL Allows the Simultaneous and Specific Visualization of Proteins, DNA Repeats, and Sites of DNA Replication.
Cytogenet Genome Res
; 159(1): 48-53, 2019.
Article
em En
| MEDLINE
| ID: mdl-31610539
ABSTRACT
Visualizing the spatiotemporal organization of the genome will improve our understanding of how chromatin structure and function are intertwined. Here, we describe a further development of the CRISPR/Cas9-based RNA-guided endonuclease-in situ labeling (RGEN-ISL) method. RGEN-ISL allowed the differentiation between vertebrate-type (TTAGGG)n and Arabidopsis-type (TTTAGGG)n telomere repeats. Using maize as an example, we established a combination of RGEN-ISL, immunostaining, and EdU labeling to visualize in situ specific repeats, histone marks, and DNA replication sites, respectively. The effects of the non-denaturing RGEN-ISL and standard denaturing FISH on the chromatin structure were compared using super-resolution microscopy. 3D structured illumination microscopy revealed that denaturation and acetic acid fixation impaired and flattened the chromatin. The broad range of adaptability of RGEN-ISL to different combinations of methods has the potential to advance the field of chromosome biology.
Palavras-chave
Texto completo:
1
Base de dados:
MEDLINE
Assunto principal:
Arabidopsis
/
Zea mays
/
Replicação do DNA
/
Sistemas CRISPR-Cas
/
Amaryllidaceae
Idioma:
En
Revista:
Cytogenet Genome Res
Assunto da revista:
GENETICA
Ano de publicação:
2019
Tipo de documento:
Article