Unraveling the functional role of DNA demethylation at specific promoters by targeted steric blockage of DNA methyltransferase with CRISPR/dCas9.
Animals
CRISPR-Cas Systems
/ genetics
CpG Islands
/ genetics
DNA Demethylation
DNA Methylation
DNA Modification Methylases
/ genetics
Epigenesis, Genetic
Fragile X Mental Retardation Protein
/ genetics
Gene Editing
/ methods
HEK293 Cells
Humans
Mice
Mixed Function Oxygenases
/ genetics
NIH 3T3 Cells
Promoter Regions, Genetic
/ genetics
Proto-Oncogene Proteins
/ genetics
RNA, Guide, Kinetoplastida
/ metabolism
Serpins
/ genetics
Journal
Nature communications
ISSN: 2041-1723
Titre abrégé: Nat Commun
Pays: England
ID NLM: 101528555
Informations de publication
Date de publication:
29 09 2021
29 09 2021
Historique:
received:
31
03
2020
accepted:
07
09
2021
entrez:
30
9
2021
pubmed:
1
10
2021
medline:
24
10
2021
Statut:
epublish
Résumé
Despite four decades of research to support the association between DNA methylation and gene expression, the causality of this relationship remains unresolved. Here, we reaffirm that experimental confounds preclude resolution of this question with existing strategies, including recently developed CRISPR/dCas9 and TET-based epigenetic editors. Instead, we demonstrate a highly effective method using only nuclease-dead Cas9 and guide RNA to physically block DNA methylation at specific targets in the absence of a confounding flexibly-tethered enzyme, thereby enabling the examination of the role of DNA demethylation per se in living cells, with no evidence of off-target activity. Using this method, we probe a small number of inducible promoters and find the effect of DNA demethylation to be small, while demethylation of CpG-rich FMR1 produces larger changes in gene expression. This method could be used to reveal the extent and nature of the contribution of DNA methylation to gene regulation.
Identifiants
pubmed: 34588447
doi: 10.1038/s41467-021-25991-9
pii: 10.1038/s41467-021-25991-9
pmc: PMC8481236
doi:
Substances chimiques
FMR1 protein, human
0
Proto-Oncogene Proteins
0
RNA, Guide
0
SERPIN-B5
0
Serpins
0
Fragile X Mental Retardation Protein
139135-51-6
Mixed Function Oxygenases
EC 1.-
TET1 protein, human
EC 1.-
DNA Modification Methylases
EC 2.1.1.-
Types de publication
Journal Article
Research Support, Non-U.S. Gov't
Langues
eng
Sous-ensembles de citation
IM
Pagination
5711Subventions
Organisme : CIHR
ID : PJT-159583
Pays : Canada
Informations de copyright
© 2021. The Author(s).
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