Details

Autor(en) / Beteiligte

• Li, Jiexin
• Chen, Zhuojia
• Chen, Feng
• Xie, Guoyou
• Ling, Yuyi
• Peng, Yanxi
• Lin, Yu
• Luo, Nan
• Chiang, Cheng-Ming
• Wang, Hongsheng

Titel

Targeted mRNA demethylation using an engineered dCas13b-ALKBH5 fusion protein

Ist Teil von

• Nucleic acids research, 2020-06, Vol.48 (10), p.5684-5694

Ort / Verlag

England

Erscheinungsjahr

2020

Quelle

MEDLINE

Beschreibungen/Notizen

• 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.