Enhanced CRISPR-based DNA demethylation by Casilio-ME-mediated RNA-guided coupling of methylcytosine oxidation and DNA repair pathways.
5-Methylcytosine
/ metabolism
CRISPR-Cas Systems
Cell Cycle Proteins
/ metabolism
Cell Line, Tumor
Cell Proliferation
Clustered Regularly Interspaced Short Palindromic Repeats
DNA Demethylation
DNA Glycosylases
/ metabolism
DNA Methylation
DNA Repair
DNA-(Apurinic or Apyrimidinic Site) Lyase
/ metabolism
Gene Editing
HEK293 Cells
Humans
Mixed Function Oxygenases
/ genetics
Oxidation-Reduction
Promoter Regions, Genetic
Proto-Oncogene Proteins
/ genetics
RNA, Guide, Kinetoplastida
/ metabolism
Sequence Analysis, RNA
Journal
Nature communications
ISSN: 2041-1723
Titre abrégé: Nat Commun
Pays: England
ID NLM: 101528555
Informations de publication
Date de publication:
20 09 2019
20 09 2019
Historique:
received:
20
05
2019
accepted:
04
09
2019
entrez:
22
9
2019
pubmed:
22
9
2019
medline:
7
1
2020
Statut:
epublish
Résumé
Here we develop a methylation editing toolbox, Casilio-ME, that enables not only RNA-guided methylcytosine editing by targeting TET1 to genomic sites, but also by co-delivering TET1 and protein factors that couple methylcytosine oxidation to DNA repair activities, and/or promote TET1 to achieve enhanced activation of methylation-silenced genes. Delivery of TET1 activity by Casilio-ME1 robustly alters the CpG methylation landscape of promoter regions and activates methylation-silenced genes. We augment Casilio-ME1 to simultaneously deliver the TET1-catalytic domain and GADD45A (Casilio-ME2) or NEIL2 (Casilio-ME3) to streamline removal of oxidized cytosine intermediates to enhance activation of targeted genes. Using two-in-one effectors or modular effectors, Casilio-ME2 and Casilio-ME3 remarkably boost gene activation and methylcytosine demethylation of targeted loci. We expand the toolbox to enable a stable and expression-inducible system for broader application of the Casilio-ME platforms. This work establishes a platform for editing DNA methylation to enable research investigations interrogating DNA methylomes.
Identifiants
pubmed: 31541098
doi: 10.1038/s41467-019-12339-7
pii: 10.1038/s41467-019-12339-7
pmc: PMC6754513
doi:
Substances chimiques
Cell Cycle Proteins
0
GADD45A protein, human
0
Proto-Oncogene Proteins
0
RNA, Guide
0
5-Methylcytosine
6R795CQT4H
Mixed Function Oxygenases
EC 1.-
TET1 protein, human
EC 1.-
DNA Glycosylases
EC 3.2.2.-
DNA-(Apurinic or Apyrimidinic Site) Lyase
EC 4.2.99.18
NEIL2 protein, human
EC 4.2.99.18
Types de publication
Journal Article
Research Support, N.I.H., Extramural
Research Support, Non-U.S. Gov't
Langues
eng
Sous-ensembles de citation
IM
Pagination
4296Subventions
Organisme : NCI NIH HHS
ID : P30 CA034196
Pays : United States
Organisme : NCI NIH HHS
ID : R01 CA115783
Pays : United States
Organisme : NHGRI NIH HHS
ID : R01 HG009900
Pays : United States
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