Targeted mRNA demethylation using an engineered dCas13b-ALKBH5 fusion protein.
5' Untranslated Regions
Adenosine
/ analogs & derivatives
AlkB Homolog 5, RNA Demethylase
/ genetics
CRISPR-Associated Proteins
/ genetics
CRISPR-Cas Systems
Cell Proliferation
Demethylation
HEK293 Cells
HeLa Cells
Humans
Oncogenes
Prevotella
/ enzymology
Protein Engineering
RNA, Messenger
/ chemistry
Recombinant Fusion Proteins
/ metabolism
Journal
Nucleic acids research
ISSN: 1362-4962
Titre abrégé: Nucleic Acids Res
Pays: England
ID NLM: 0411011
Informations de publication
Date de publication:
04 06 2020
04 06 2020
Historique:
accepted:
08
04
2020
revised:
01
04
2020
received:
22
11
2019
pubmed:
2
5
2020
medline:
18
8
2020
entrez:
2
5
2020
Statut:
ppublish
Résumé
Studies on biological functions of N6-methyladenosine (m6A) modification in mRNA have drawn significant attention in recent years. Here we describe the construction and characterization of a CRISPR-Cas13b-based tool for targeted demethylation of specific mRNA. A fusion protein, named dm6ACRISPR, was created by linking a catalytically inactive Type VI-B Cas13 enzyme from Prevotella sp. P5-125 (dPspCas13b) to m6A demethylase AlkB homolog 5 (ALKBH5). dm6ACRISPR specifically demethylates m6A of targeted mRNA such as cytochrome b5 form A (CYB5A) to increase its mRNA stability. It can also demethylate β-catenin-encoding CTNNB1 mRNA that contains multiple m6A sites to trigger its translation. In addition, the dm6ACRISPR system incurs efficient demethylation of targeted epitranscriptome transcripts with limited off-target effects. Targeted demethylation of transcripts coding for oncoproteins such as epidermal growth factor receptor (EGFR) and MYC can suppress proliferation of cancer cells. Together, we provide a programmable and in vivo manipulation tool to study mRNA modification of specific genes and their related biological functions.
Identifiants
pubmed: 32356894
pii: 5827664
doi: 10.1093/nar/gkaa269
pmc: PMC7261189
doi:
Substances chimiques
5' Untranslated Regions
0
CRISPR-Associated Proteins
0
RNA, Messenger
0
Recombinant Fusion Proteins
0
N-methyladenosine
CLE6G00625
AlkB Homolog 5, RNA Demethylase
EC 1.14.11.-
Adenosine
K72T3FS567
Types de publication
Journal Article
Research Support, N.I.H., Extramural
Research Support, Non-U.S. Gov't
Research Support, U.S. Gov't, Non-P.H.S.
Langues
eng
Sous-ensembles de citation
IM
Pagination
5684-5694Subventions
Organisme : NCI NIH HHS
ID : R01 CA251698
Pays : United States
Informations de copyright
© The Author(s) 2020. Published by Oxford University Press on behalf of Nucleic Acids Research.
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