Live-cell imaging of genomic loci with Cas9 variants.
CRISPR/Cas9
dCas9 variants
dynamic changes
live-cell imaging
Journal
Biotechnology journal
ISSN: 1860-7314
Titre abrégé: Biotechnol J
Pays: Germany
ID NLM: 101265833
Informations de publication
Date de publication:
Dec 2022
Dec 2022
Historique:
revised:
14
08
2022
received:
20
07
2021
accepted:
22
08
2022
pubmed:
5
9
2022
medline:
25
2
2023
entrez:
4
9
2022
Statut:
ppublish
Résumé
Endonuclease-deactivated clustered regularly interspaced short palindromic repeats (CRISPR)-associated nuclease (dCas9) has been repurposed for live-cell imaging of genomic loci. Engineered or evolved dCas9 variants have been developed to increase the applicability of the CRISPR/dCas9 system. However, there have been no systematic comparisons of these dCas9 variants in terms of their performance in the visualization of genomic loci. Here we demonstrate that dSpCas9 and its variants deSpCas9(1.1), dSpCas9-HF1, devoCas9, and dxCas9(3.7) can be used for CRISPR-based live-cell genomic imaging. dSpCas9 had the greatest utility, with a high labeling efficiency of repetitive sequences-including those with a low number of repeats-and good compatibility with target RNA sequences at the MUC4 locus that varied in length from 13 to 23 nucleotides. We combined CRISPR-Tag with the dSpCas9 imaging system to observe the dynamics of the Tet promoter and found that its movement was restricted when it was active. These novel Cas9 variants provide a new set of tools for investigating the spatiotemporal regulation of gene expression through live imaging of genomic sites.
Sections du résumé
BACKGROUND
BACKGROUND
Endonuclease-deactivated clustered regularly interspaced short palindromic repeats (CRISPR)-associated nuclease (dCas9) has been repurposed for live-cell imaging of genomic loci. Engineered or evolved dCas9 variants have been developed to increase the applicability of the CRISPR/dCas9 system. However, there have been no systematic comparisons of these dCas9 variants in terms of their performance in the visualization of genomic loci.
MAIN METHODS AND MAJOR RESULTS
RESULTS
Here we demonstrate that dSpCas9 and its variants deSpCas9(1.1), dSpCas9-HF1, devoCas9, and dxCas9(3.7) can be used for CRISPR-based live-cell genomic imaging. dSpCas9 had the greatest utility, with a high labeling efficiency of repetitive sequences-including those with a low number of repeats-and good compatibility with target RNA sequences at the MUC4 locus that varied in length from 13 to 23 nucleotides. We combined CRISPR-Tag with the dSpCas9 imaging system to observe the dynamics of the Tet promoter and found that its movement was restricted when it was active.
CONCLUSIONS AND IMPLICATIONS
CONCLUSIONS
These novel Cas9 variants provide a new set of tools for investigating the spatiotemporal regulation of gene expression through live imaging of genomic sites.
Identifiants
pubmed: 36058644
doi: 10.1002/biot.202100381
doi:
Substances chimiques
Endonucleases
EC 3.1.-
RNA
63231-63-0
Types de publication
Journal Article
Langues
eng
Sous-ensembles de citation
IM
Pagination
e2100381Subventions
Organisme : the National Natural Science Foundation of China
ID : 82072329
Organisme : the National Natural Science Foundation of China
ID : 82070002
Organisme : the National Natural Science Foundation of China
ID : 81872511
Organisme : National Science and Technology Major Project
ID : 2018ZX10301101
Organisme : Frontier Research Program of Bioland Laboratory
ID : 2018GZR110105005
Informations de copyright
© 2022 Wiley-VCH GmbH.
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