CRISPR/Cas9-Based RGEN-ISL Allows the Simultaneous and Specific Visualization of Proteins, DNA Repeats, and Sites of DNA Replication

Kód výsledku v IS VaVaI

<a href="https://www.isvavai.cz/riv?ss=detail&h=RIV%2F61389030%3A_____%2F19%3A00517551" target="_blank" >RIV/61389030:_____/19:00517551 - isvavai.cz</a>

Výsledek na webu

<a href="http://dx.doi.org/10.1159/000502600" target="_blank" >http://dx.doi.org/10.1159/000502600</a>

DOI - Digital Object Identifier

<a href="http://dx.doi.org/10.1159/000502600" target="_blank" >10.1159/000502600</a>

Jazyk výsledku

angličtina

Název v původním jazyce

CRISPR/Cas9-Based RGEN-ISL Allows the Simultaneous and Specific Visualization of Proteins, DNA Repeats, and Sites of DNA Replication

Popis výsledku v původním jazyce

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.

Název v anglickém jazyce

CRISPR/Cas9-Based RGEN-ISL Allows the Simultaneous and Specific Visualization of Proteins, DNA Repeats, and Sites of DNA Replication

Popis výsledku anglicky

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.

Druh

J_imp - Článek v periodiku v databázi Web of Science

CEP obor

—

OECD FORD obor

10603 - Genetics and heredity (medical genetics to be 3)

Projekt

<a href="/cs/project/GA17-14048S" target="_blank" >GA17-14048S: Časová a prostorová charakterizace replikace příbuzných rostlinných druhů s kontrastní velikostí genomů</a><br>

Návaznosti

I - Institucionalni podpora na dlouhodoby koncepcni rozvoj vyzkumne organizace

Rok uplatnění

2019

Kód důvěrnosti údajů

S - Úplné a pravdivé údaje o projektu nepodléhají ochraně podle zvláštních právních předpisů

Název periodika

Cytogenetic and Genome Research

ISSN

1424-8581

e-ISSN

—

Svazek periodika

159

Číslo periodika v rámci svazku

1

Stát vydavatele periodika

CH - Švýcarská konfederace

Počet stran výsledku

6

Strana od-do

48-53

Kód UT WoS článku

000498640000007

EID výsledku v databázi Scopus

2-s2.0-85074473340