Simple and versatile imaging of genomic loci in live mammalian cells and early pre-implantation embryos using CAS-LiveFISH

Abstract Visualizing the 4D genome in live cells is essential for understanding its regulation. Programmable DNA-binding probes, such as fluorescent clustered regularly interspaced short palindromic repeats (CRISPR) and transcription activator-like effector (TALE) proteins have recently emerged as p...

Descripción completa

Guardado en:

Detalles Bibliográficos
Autores principales:	Yongtao Geng, Alexandros Pertsinidis
Formato:	article
Lenguaje:	EN
Publicado:	Nature Portfolio 2021
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/69f10d4b7e3545b2b72262644ef6d135
Etiquetas:	Agregar Etiqueta Sin Etiquetas, Sea el primero en etiquetar este registro!

id	oai:doaj.org-article:69f10d4b7e3545b2b72262644ef6d135
record_format	dspace
spelling	oai:doaj.org-article:69f10d4b7e3545b2b72262644ef6d1352021-12-02T15:02:51ZSimple and versatile imaging of genomic loci in live mammalian cells and early pre-implantation embryos using CAS-LiveFISH10.1038/s41598-021-91787-y2045-2322https://doaj.org/article/69f10d4b7e3545b2b72262644ef6d1352021-06-01T00:00:00Zhttps://doi.org/10.1038/s41598-021-91787-yhttps://doaj.org/toc/2045-2322Abstract Visualizing the 4D genome in live cells is essential for understanding its regulation. Programmable DNA-binding probes, such as fluorescent clustered regularly interspaced short palindromic repeats (CRISPR) and transcription activator-like effector (TALE) proteins have recently emerged as powerful tools for imaging specific genomic loci in live cells. However, many such systems rely on genetically-encoded components, often requiring multiple constructs that each must be separately optimized, thus limiting their use. Here we develop efficient and versatile systems, based on in vitro transcribed single-guide-RNAs (sgRNAs) and fluorescently-tagged recombinant, catalytically-inactivated Cas9 (dCas9) proteins. Controlled cell delivery of pre-assembled dCas9-sgRNA ribonucleoprotein (RNP) complexes enables robust genomic imaging in live cells and in early mouse embryos. We further demonstrate multiplex tagging of up to 3 genes, tracking detailed movements of chromatin segments and imaging spatial relationships between a distal enhancer and a target gene, with nanometer resolution in live cells. This simple and effective approach should facilitate visualizing chromatin dynamics and nuclear architecture in various living systems.Yongtao GengAlexandros PertsinidisNature PortfolioarticleMedicineRScienceQENScientific Reports, Vol 11, Iss 1, Pp 1-11 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Yongtao Geng Alexandros Pertsinidis Simple and versatile imaging of genomic loci in live mammalian cells and early pre-implantation embryos using CAS-LiveFISH
description	Abstract Visualizing the 4D genome in live cells is essential for understanding its regulation. Programmable DNA-binding probes, such as fluorescent clustered regularly interspaced short palindromic repeats (CRISPR) and transcription activator-like effector (TALE) proteins have recently emerged as powerful tools for imaging specific genomic loci in live cells. However, many such systems rely on genetically-encoded components, often requiring multiple constructs that each must be separately optimized, thus limiting their use. Here we develop efficient and versatile systems, based on in vitro transcribed single-guide-RNAs (sgRNAs) and fluorescently-tagged recombinant, catalytically-inactivated Cas9 (dCas9) proteins. Controlled cell delivery of pre-assembled dCas9-sgRNA ribonucleoprotein (RNP) complexes enables robust genomic imaging in live cells and in early mouse embryos. We further demonstrate multiplex tagging of up to 3 genes, tracking detailed movements of chromatin segments and imaging spatial relationships between a distal enhancer and a target gene, with nanometer resolution in live cells. This simple and effective approach should facilitate visualizing chromatin dynamics and nuclear architecture in various living systems.
format	article
author	Yongtao Geng Alexandros Pertsinidis
author_facet	Yongtao Geng Alexandros Pertsinidis
author_sort	Yongtao Geng
title	Simple and versatile imaging of genomic loci in live mammalian cells and early pre-implantation embryos using CAS-LiveFISH
title_short	Simple and versatile imaging of genomic loci in live mammalian cells and early pre-implantation embryos using CAS-LiveFISH
title_full	Simple and versatile imaging of genomic loci in live mammalian cells and early pre-implantation embryos using CAS-LiveFISH
title_fullStr	Simple and versatile imaging of genomic loci in live mammalian cells and early pre-implantation embryos using CAS-LiveFISH
title_full_unstemmed	Simple and versatile imaging of genomic loci in live mammalian cells and early pre-implantation embryos using CAS-LiveFISH
title_sort	simple and versatile imaging of genomic loci in live mammalian cells and early pre-implantation embryos using cas-livefish
publisher	Nature Portfolio
publishDate	2021
url	https://doaj.org/article/69f10d4b7e3545b2b72262644ef6d135
work_keys_str_mv	AT yongtaogeng simpleandversatileimagingofgenomiclociinlivemammaliancellsandearlypreimplantationembryosusingcaslivefish AT alexandrospertsinidis simpleandversatileimagingofgenomiclociinlivemammaliancellsandearlypreimplantationembryosusingcaslivefish
_version_	1718389082706411520

Simple and versatile imaging of genomic loci in live mammalian cells and early pre-implantation embryos using CAS-LiveFISH

Ejemplares similares